Processes for immobilising biological entities

ABSTRACT

According to the invention there is provided inter alia a process for the manufacture of a solid object having a surface comprising a layered coating of cationic and anionic polymer wherein the outer coating layer comprises an anticoagulant entity, comprising the steps of: 
     i) treating a surface of the solid object with a cationic polymer;
 
ii) treating the surface with an anionic polymer;
 
iii) optionally repeating steps i) and ii) one or more times;
 
iv) treating the surface with a cationic polymer; and
 
v) treating the outermost layer of cationic polymer with an anticoagulant entity, thereby to covalently attach the anticoagulant entity to the outermost layer of cationic polymer; wherein, the anionic polymer is characterized by having (a) a total molecular weight of 650 kDa-10,000 kDa; and (b) a solution charge density of &gt;4 μeq/g; and wherein, step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

FIELD OF THE INVENTION

The present invention relates to processes for preparing solid objectshaving surface coatings comprising biological entities. In particular,the present invention relates to processes for preparing improvedsurface coatings comprising anticoagulant entities such as heparin andcertain products obtained thereby.

BACKGROUND OF THE INVENTION

When a medical device is implanted in the body or is in contact withbody fluids, a number of different reactions are set into motion, someof them resulting in inflammation and some in the coagulation of theblood in contact with the device surface. In order to counteract theseserious adverse effects, the well-known anticoagulant compound heparinhas for a long time been administered systemically to patients beforethe medical device is implanted into their body, or when it is incontact with their body fluids, in order to provide an antithromboticeffect.

Thrombin is one of several coagulation factors, all of which worktogether to result in the formation of thrombi at a surface in contactwith the blood. Antithrombin (also known as antithrombin III) (“ATIII”)is the most prominent coagulation inhibitor. It neutralizes the actionof thrombin and other coagulation factors and thus restricts or limitsblood coagulation. Heparin dramatically enhances the rate at whichantithrombin inhibits coagulation factors. Heparin cofactor II (“HCII”)is another coagulation factor which rapidly inhibits thrombin in thepresence of heparin.

Systemic treatment with high doses of heparin is, however, oftenassociated with serious side-effects of which bleeding is thepredominant. Another rare, but serious complication of heparin therapyis the development of an allergic response called heparin inducedthrombocytopenia (HIT) that may lead to thrombosis (both venous andarterial). High-dose systemic heparin treatment e.g. during surgery alsorequires frequent monitoring of the activated clotting time (used tomonitor and guide heparin therapy) and the corresponding doseadjustments as necessary.

Therefore, solutions have been sought where the need for a systemicheparinization of the patient would be unnecessary or can be limited. Itwas thought that this could be achieved through a surface modificationof the medical devices using the anticoagulative properties of heparinand other anticoagulants. Thus, a number of more or less successfultechnologies have been developed where a layer of heparin is attached tothe surface of the medical device seeking thereby to render the surfacethromboresistant. For devices where long-term bioactivity is required,heparin should desirably be resistant to leaching and degradation.

Heparin is a polysaccharide carrying negatively charged sulfate andcarboxylic acid groups on the saccharide units. Ionic binding of heparinto polycationic surfaces was thus attempted, but the surfacemodifications suffered from lack of stability resulting in lack offunction, as the heparin leached from the surface. Thereafter, differentsurface modifications have been prepared wherein the heparin has beencovalently bound to groups on the surface.

One of the most successful processes for rendering a medical devicethromboresistant has been the covalent binding of a heparin fragment toa modified surface of the device. The general method and improvementsthereof are described in various patent documents (see EP0086186A1,EP0086187A1, EP0495820B1 and U.S. Pat. No. 6,461,665B1 each of which isincorporated herein by reference in its entirety).

These documents describe the preparation of a heparinized surface byreacting heparin modified to bear a terminal aldehyde group with asurface on a medical device which has been modified to bear primaryamino groups. An intermediate Schiff base is formed which is reduced insitu to form a stable secondary amine linker, thereby covalentlyimmobilizing the heparin.

Further methods for covalently attaching heparin to a surface whileretaining its activity are described in WO2010/029189A2, WO2011/110684A1and WO2012/123384A1 (each of which is incorporated herein by referencein its entirety).

The anticoagulant entity is typically immobilized on a surface which hasbeen treated with one or more layers of polymer or a complex, ratherthan being immobilized directly onto the surface of the solid object.

EP0086187A1 describes a surface modified substrate with a complexabsorbed thereto, wherein the complex is of a polymeric cationicsurfactant that contains primary amino nitrogen functionality as well assecondary and/or tertiary amino functionality, and a dialdehyde that has1-4 carbon atoms between the two aldehyde groups. An anionic compoundmay additionally be bonded to said complex, and optionally additionalcationic and anionic alternating compounds.

EP0495820B1 describes a method for modifying the surface of a substrate,comprising the steps of: (a) adsorbing a polyamine of a high averagemolecular weight and crosslinking said polyamine with crotonaldehyde;(b) then adsorbing on the surface of the crosslinked polyamine a layerof an anionic polysaccharide; (c) optionally repeating steps (a) and (b)one or more times; and (d) adsorbing on the anionic polysaccharidelayer, or on the outermost layer of anionic polysaccharide, a layer ofnon-crosslinked polyamine providing free primary amino groups. In asubsequent step, a biologically active chemical entity carrying afunctional group reactive with the free primary amino groups can bebound to the non-crosslinked polyamine, e.g. heparin.

However, there remains a need for improved surface coatings comprisinganticoagulant entities such as heparin, in particular for coatings inwhich the biological activity of the anticoagulant entity is maintainedor enhanced. Such improved surface coatings have potential utility inmedical devices and other articles which would benefit from ananticoagulant surface.

The present inventors have discovered that, surprisingly, the nature ofthe surface upon which an anticoagulant entity is immobilized cansignificantly impact characteristics of the coating, in particular theresulting biological activity of the anticoagulant entity. Inparticular, when an anticoagulant entity is immobilized on a surface ofa solid object comprising a layered coating of cationic and anionicpolymer, careful modulation of the nature and the conditions of theapplication of the anionic polymer layer(s) can improve the resultingcharacteristics of the coating of the solid object including, forexample, the thromboresistant properties that it may have.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a process for themanufacture of a solid object having a surface comprising a layeredcoating of cationic and anionic polymer wherein the outer coating layercomprises an anticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer; wherein,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a solution charge density of >4μeq/g;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In a further aspect, the present invention provides a process for themanufacture of a solid object having a surface comprising a layeredcoating of cationic and anionic polymer wherein the outer coating layercomprises an anticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is a polymer comprising —SO₃ ⁻ groups,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a sulfur content between 10% and25% by weight of the anionic polymer;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In a further aspect, the present invention provides a process for themanufacture of a solid object having a surface comprising a layeredcoating of cationic and anionic polymer wherein the outer coating layercomprises an anticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is characterized by having a total molecular weightof 650 kDa-10,000 kDa;

the anionic polymer is dextran sulfate;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: shows an example coating of the invention with a single bilayer;

FIG. 2: shows normalized heparin activity (HA) for PVC tubing coatedwith dextran sulfates 4, 5, 6 and 7 at 0.25 M and 1.7 M NaClconcentration;

FIG. 3: shows normalized heparin activity (HA) for PVC tubing coatedwith dextran sulfate 5 using different salts at different concentration;

FIG. 4: shows heparin concentration (HC) for PVC tubing coated withdextran sulfates 3, 4, 5, 6 and 7 at 1.7 M NaCl concentration;

FIG. 5: shows heparin concentration (HC) for PVC tubing coated withdextran sulfates 3, 4, 5, 6 and 7 at varied NaCl concentration;

FIG. 6: shows heparin concentration (HC) for PVC tubing coated withdextran sulfate 5 using different salts at different concentration;

FIG. 7: shows zeta potential for PVC tubing coated with dextran sulfates3, 4 and 5 at 1.7 M NaCl concentration;

FIG. 8: shows zeta potential for PVC tubing coated with dextran sulfates3, 6 and 7 at 1.7 M NaCl concentration;

FIG. 9: shows zeta potential for PVC tubing coated with dextran sulfates3, 4 and 5 at 0.25 M NaCl concentration;

FIG. 10: shows zeta potential for PVC tubing coated with dextransulfates 3, 6 and 7 at 0.25 M NaCl concentration;

FIG. 11: shows zeta potential for PVC tubing coated with dextran sulfate5 at varied NaCl concentration;

FIG. 12: shows zeta potential for PVC tubing coated with dextran sulfate5 at varied Na₂HPO₄ concentration;

FIG. 13: shows zeta potential for PVC tubing coated with dextran sulfate5 at varied Na₂SO₄ concentration;

FIG. 14: shows preserved platelets (%) for PVC tubing coated withdextran sulfates 2, 4, 5, 6 and 7 at 0.25 M NaCl concentration;

FIG. 15: shows F1+2 (prothrombin fragment) for PVC tubing coated withdextran sulfates 2, 4, 5, 6 and 7 at 0.25 M NaCl concentration;

FIG. 16: shows preserved platelets (%) for PVC tubing coated withdextran sulfates 4, 5, 6 and 7 at 1.7 M NaCl concentration;

FIG. 17: shows F1+2 (prothrombin fragment) for PVC tubing coated withdextran sulfates 4, 5, 6 and 7 at 1.7 M NaCl concentration;

FIG. 18: shows preserved platelets (%) for PVC tubing coated withdextran sulfate 4 at 0.25 M NaCl concentration, pre- andpost-temperature and humidity test;

FIG. 19: shows F1+2 (prothrombin fragment) for PVC tubing coated withdextran sulfate 4 at 0.25 M NaCl concentration, pre- andpost-temperature and humidity test;

FIG. 20: shows preserved platelets (%) for PVC tubing coated withdextran sulfates 4, 5 and 7 at 1.7 M NaCl concentration, pre- andpost-temperature and humidity test;

FIG. 21: shows F1+2 (prothrombin fragment) for PVC tubing coated withdextran sulfates 4, 5 and 7 at 1.7 M NaCl concentration, pre- andpost-temperature and humidity test;

FIG. 22: shows a typical zeta potential profile of a solid object of thepresent invention.

FIG. 23: shows the active pentasaccharide sequence of heparin

DETAILED DESCRIPTION OF THE INVENTION

Solid Object

Any solid object can potentially be coated using the process of theinvention, although such coatings and processes are particularly usefulfor medical devices, analytical devices, separation devices, and otherindustrial articles including membranes.

Solid objects may have a thromboresistant surface. In certainembodiments of the invention, the thromboresistant surface may exhibit adirect pharmacologic inhibition of the coagulation response byimmobilization of an anticoagulant entity. In certain embodiments of theinvention, the thromboresistant surface does not cause any appreciableclinically-significant adverse reactions such as thrombosis, haemolysis,platelet, leukocyte, and complement activation, and/or otherblood-associated adverse event when in contact with blood.

In the art, the terms “hemocompatible”, “non-thrombogenic”,“anti-thrombogenic” and the like can typically be interpreted as beingequivalent to the term “thromboresistant”.

In one embodiment, the solid object is a medical device. When the solidobject is a medical device, it is suitably a thromboresistant medicaldevice. Thus, in one embodiment the solid object is a thromboresistantmedical device. For the purposes of this patent application, the term“medical device” refers to intracorporeal or extra-corporeal devices butmore usually to intracorporeal medical devices.

Intracorporeal medical devices are devices which are used within theanatomy e.g. within the vasculature or other body lumen, space orcavity, typically to provide a therapeutic effect. Intracorporealdevices may be of long-term or temporary use. Devices of long-term useare left, in part or in whole, in the anatomy after the immediatesurgical procedure to deliver them e.g. stents or stent-grafts. Devicesfor temporary or short-term use include those which are transientlyinserted into a treatment region (i.e. inserted and then removed in thesame surgical procedure), such as a medical balloon. In one embodiment,the solid object is an intracorporeal medical device.

Examples of intracorporeal medical devices which can be permanent ortemporary intracorporeal medical devices include stents includingbifurcated stents, balloon-expandable stents, self-expanding stents,neurovascular stents and flow diverting stents, stent-grafts includingbifurcated stent-grafts, grafts including vascular grafts and bifurcatedgrafts, sheaths including retractable sheaths such as interventionaldiagnostic and therapeutic sheaths, large and standard bore endovasculardelivery sheaths, arterial introducer sheaths with and withouthemostatic control and with or without steering, micro-introducersheaths, dialysis access sheaths, guiding sheaths, and percutaneoussheaths, dilators, occluders such as vascular occluders, embolicfilters, embolectomy devices, catheters, artificial blood vessels, bloodindwelling monitoring devices, valves including artificial heart valves,pacemaker electrodes, guidewires, cardiac leads, cardiopulmonary bypasscircuits, cannulae, plugs, drug delivery devices, balloons, tissue patchdevices, blood pumps, patches, lines such as chronic infusion lines orarterial lines, placement wires, devices for continuous subarachnoidinfusions, feeding tubes, CNS shunts such as ventriculopleural shunts,ventriculoatrial (VA) shunts, ventriculoperitoneal (VP) shunts,ventricular atrial shunts, portosystemic shunts and shunts for ascites.

Examples of catheters include, but are not limited to, microcatheters,central venous catheters, peripheral intravenous catheters, hemodialysiscatheters, catheters such as coated catheters include implantable venouscatheters, tunnelled venous catheters, coronary catheters useful forangiography, angioplasty, or ultrasound procedures in the heart or inperipheral veins and arteries, catheters containing spectroscopic orimaging capabilities, hepatic artery infusion catheters, CVC (centralvenous catheters), peripheral intravenous catheters, peripherallyinserted central venous catheters (PIC lines), flow-directedballoon-tipped pulmonary artery catheters, total parenteral nutritioncatheters, chronic dwelling catheters (e.g. chronic dwellinggastrointestinal catheters and chronic dwelling genitourinarycatheters), peritoneal dialysis catheters, CPB catheters(cardiopulmonary bypass), urinary catheters and microcatheters (e.g. forintracranial application).

In one embodiment, the solid object is an intracorporeal medical deviceselected from the group consisting of stents, stent-grafts, sheaths,dilators, occluders, valves, embolic filters, embolectomy devices,catheters, artificial blood vessels, blood indwelling monitoringdevices, valves, pacemaker electrodes, guidewires, cardiac leads,cardiopulmonary bypass circuits, cannulae, plugs, drug delivery devices,balloons, tissue patch devices, blood pumps, patches, lines, placementwires, devices for continuous subarachnoid infusions, feeding tubes andshunts. In a specific embodiment, the solid object is a stent or astent-graft.

In one embodiment, said intracorporeal medical device can be used inneurological, peripheral, cardiac, orthopaedic, dermal, or gynaecologicapplications. In one embodiment, said stents can be used in cardiac,peripheral or neurological applications. In one embodiment, saidstent-grafts can be used in cardiac, peripheral or neurologicalapplications. In one embodiment, said sheaths can be used in carotid,renal, transradial, transseptal, paediatric or micro applications.

Examples of extracorporeal medical devices are blood treatment devices,and transfusion devices. In one embodiment, said intracorporeal medicaldevice can be used in neurological, peripheral, cardiac, orthopaedic,dermal, or gynaecologic applications. In one embodiment theextracorporeal medical device is an oxygenator. In another embodimentthe extracorporeal medical device is a filter capable of removingviruses, bacteria, sepsis-causing pro-inflammatory cytokines and toxins.

A membrane can be, for example, a haemodialysis membrane.

An analytical device can be, for example, a solid support for carryingout an analytical process such as chromatography or an immunologicalassay, reactive chemistry or catalysis. Examples of such devices includeslides, beads, well plates and membranes.

A separation device can be, for example, a solid support for carryingout a separation process such as protein purification, affinitychromatography or ion exchange. Examples of such devices include filtersand columns.

The solid object may comprise or be formed of a metal, a synthetic ornaturally occurring organic or inorganic polymer, a ceramic material, aprotein-based material, or a polysaccharide-based material, inter alia.

Suitable metals include, but are not limited to, biocompatible metalssuch as titanium, stainless steel, high nitrogen stainless steel,cobalt, chromium, nickel, tantalum, niobium, gold, silver, rhodium,zinc, platinum, rubidium, copper and magnesium, and combinations(alloys) thereof. Suitable alloys include cobalt-chromium alloys such asL-605, MP35N, Elgiloy, titanium alloys including nickel-titanium alloys(such as Nitinol), tantalum alloys, niobium alloys (e.g. Nb-1% Zr), andothers.

In one embodiment, said biocompatible metal is a nickel-titanium alloy,such as Nitinol.

Synthetic or naturally occurring organic or inorganic polymers includepolyolefins, polyesters (e.g. polyethylene terephthalate andpolybutylene terephthalate), polyester ethers, polyester elastomercopolymers (e.g. such as those available from DuPont in Wilmington, Del.under the tradename of HYTREL®), fluorine-containing polymers,chlorine-containing polymers (e.g. polyvinyl chloride (PVC)), blockcopolymer elastomers (e.g. such as those copolymers having styrene endblocks, and midblocks formed from butadiene, isoprene,ethylene/butylene, ethylene/propene), block copolymers (e.g. styrenicblock copolymers such as acrylonitrile-styrene andacrylonitrile-butadiene-styrene block copolymers, or block copolymerswherein the particular block copolymer thermoplastic elastomers in whichthe block copolymer is made up of hard segments of a polyester orpolyamide and soft segments of polyether), polyurethanes, polyamides(e.g. nylon 12, nylon 11, nylon 9, nylon 6/9 and nylon 6/6), polyetherblock amides (e.g. PEBAX®), polyetheresteramide, polyimides,polycarbonates, polyphenylene sulfides, polyphenylene oxides,polyethers, silicones, polycarbonates, polyhydroxyethylmethacrylate,polyvinyl pyrrolidone, polyvinyl alcohol, rubber, silicone rubber,polyhydroxyacids, polyallylamine, polyallylalcohol, polyacrylamide,polyacrylic acid, polystyrenes, polytetrafluoroethylene,poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates),poly(vinyl alcohols), polyoxymethylenes, polycarbonates, phenolics,amino-epoxy resins, cellulose-based plastics, and rubber-like plastics,bioresorbables (e.g. poly(D,L-lactide) and polyglycolids, and copolymersthereof and copolymers thereof), derivatives thereof and mixturesthereof. Combinations of these materials can be employed with andwithout cross-linking. Some of these classes are available both asthermosets and as thermoplastic polymers. As used herein, the term“copolymer” shall be used to refer to any polymer formed from two ormore monomers, e.g. 2, 3, 4, 5 and so on and so forth.

Fluorinated polymers (fluorine-containing polymers) includefluoropolymers such as expanded polytetrafluoroethylene (ePTFE),polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP),perfluorocarbon copolymers (such as tetrafluoroethyleneperfluoroalkylvinyl ether (TFE/PAVE) copolymers and copolymers oftetrafluoroethylene (TFE) and perfluoromethyl vinyl ether (PMVE)), andcombinations of the above with and without crosslinking between thepolymer chains.

In one embodiment, the solid object comprises a polyether-block-amide,such as PEBAX®. In another embodiment, the solid object comprises achlorine-containing polymer (e.g. PVC) or a fluorine-containing polymer(e.g. ePTFE).

Polymeric substrates may optionally be blended with fillers and/orcolorants. Thus, suitable substrates include pigmented materials such aspigmented polymeric materials.

Ceramic substrates may include, but are not limited to, silicone oxides,aluminium oxides, alumina, silica, hydroxyapapitites, glasses, calciumoxides, polysilanols, and phosphorous oxide.

Protein-based materials include silk and wool. Polysaccharide-basedmaterials include agarose and alginate.

Anticoagulant Entity

An anticoagulant entity is an entity capable of interacting withmammalian blood to prevent or alleviate coagulation or thrombusformation.

Anticoagulant entities include heparin moieties, dermatan sulfatemoieties, dermatan disulfate moieties, hirudin, eptifibatide,tirofibran, urokinase, D-Phe-Pro-Arg chloromethylketone, an RGDpeptide-containing compound, AZX100 (a cell peptide that mimics HSP20,Capstone Therapeutics Corp., USA), platelet receptor antagonists,anti-thrombin antibodies, anti-platelet receptor antibodies, aspirin,prostaglandin inhibitors, platelet inhibitors (e.g. clopidogrel, nitricoxide (NO), prostaglandines and abciximab), antiplatelet peptides,coumadins (i.e. vitamin K antagonists of the 4-hydroxycoumarin classe.g. warfarin), argatroban, thrombomodulin, anticoagulant proteins,anticoagulant enzymes (e.g. apyrase). In one embodiment, theanticoagulant entity is selected from the group consisting of heparinmoieties, dermatan sulfate moieties and dermatan disulfate moieties.

In one embodiment, the anticoagulant entity is a glycosaminoglycan. Inone embodiment, the anticoagulant entity is a thrombin inhibitor.

The term “heparin moiety” refers to a heparin molecule, a fragment of aheparin molecule, a derivative of a heparin molecule or an analogue of aheparin molecule.

In one embodiment, the anticoagulant entity is a heparin moiety.Suitably the heparin moiety is selected from the group consisting offull length heparin (native heparin), an alkali metal or alkaline earthmetal salt of heparin (e.g. sodium heparin (e.g. Hepsal or Pularin),potassium heparin (e.g. Clarin), lithium heparin, calcium heparin (e.g.Calciparine) or magnesium heparin (e.g. Cutheparine)), a low molecularweight heparin (e.g. ardeparin sodium, tinzaparin or dalteparin),heparan sulfate, a heparinoid, a heparin-based compound, heparin havinga hydrophobic counter-ion, a synthetic heparin composition capable ofantithrombin-mediated inhibition of factor Xa (e.g. a “fondaparinux”composition (e.g. Arixtra from GlaxoSmithKline)) and a synthetic heparinderivative comprising at least the active pentasaccharide sequence fromheparin (see for example Petitou et al., Biochimie, 2003, 85(1-2):83-9).Additional heparin moieties include heparin modified by means of e.g.mild nitrous acid degradation (U.S. Pat. No. 4,613,665A, incorporatedherein by reference in its entirety) or periodate oxidation (U.S. Pat.No. 6,653,457B1, incorporated herein by reference in its entirety) andother modification reactions known in the art where the activity of theheparin moiety is preserved. Heparin moieties also include such moietiesbound to a linker or spacer as described below. In one embodiment, theheparin moiety is full length heparin.

Low molecular weight heparins may be prepared by, for example, oxidativedepolymerisation, enzymatic degradation or deaminative cleavage.

In one embodiment the heparin moiety is a fragment of heparin. Fragmentsof heparin may be produced using techniques known in the art. Suitablythe fragments are fragments of native heparin produced by a processcomprising degrading (e.g. fragmentation of) native heparin. Asillustrated in Example 2e below, fragments of heparin may be prepared bypartial nitrous acid cleavage of native heparin, optionally followed byfractionation by gel chromatography. Alternatively, fragments of heparinmay be synthetically produced. Synthetic production may include chemoenzymatic and/or traditional organic chemistry methods.

The anticoagulant activity of heparin is mainly dependent on anantithrombin (AT) binding pentasaccharide sequence (the ‘activepentasaccharide sequence’, or ‘active sequence’; see FIG. 23). Suitablythe fragment of heparin contains the active pentasaccharide sequence.

Fragments of heparin may, for example, have a length of 5-30 e.g. 5-20e.g. 5-18 e.g. 5-17 e.g. 5-10 e.g. 6-10 saccharide residues.Alternatively, fragments of heparin may, for example, have a length of6-30 e.g. 6-20 e.g. 6-18 e.g. 6-17 saccharide residues.

U.S. Pat. No. 6,461,665B1 (Scholander; incorporated herein by reference)discloses improving the anti-thrombogenic activity ofsurface-immobilized heparin by treating the heparin prior toimmobilization. The improvement is achieved by treating the heparin atelevated temperature or at elevated pH, or by contacting the heparinwith nucleophilic catalysts such as amines, alcohols, thiols orimmobilized amino, hydroxyl or thiol groups.

The anticoagulant entity is covalently immobilized on the surface of thesolid object, therefore does not substantially elute or leach from thesolid object. As discussed below, the anticoagulant entity can becovalently immobilized by various methods.

The anticoagulant entity is covalently attached to the outermost layerof cationic polymer.

The anticoagulant entity is suitably end-point attached to the cationicpolymer, particularly when the anticoagulant entity is a heparin moiety.Thus, in one embodiment, the anticoagulant entity is an end-pointattached anticoagulant moiety. In a particular embodiment, theanticoagulant entity is an end-point attached heparin moiety. Whereapplicable, the anticoagulant entity is preferably connected through itsreducing end. Thus, in one embodiment, the anticoagulant entity isconnected through its reducing end. In a particular embodiment, theanticoagulant entity is an end-point attached heparin moiety connectedthrough its reducing end (sometimes referred to as position C1 of thereducing terminal). The advantage of end-point attachment, especiallyreducing end-point attachment, is that the biological activity of theanticoagulant entity (for example the heparin moiety) is maximized dueto enhanced availability e.g. the antithrombin interaction sites ascompared with attachment elsewhere in the anticoagulant entity (e.g.heparin moiety).

A representative end-point attachment process is described inEP0086186B1 (Larm; incorporated herein by reference in its entirety)which discloses a process for the covalent binding of oligomeric orpolymeric organic substances to substrates of different types containingprimary amino groups. The substance to be coupled, which may be heparin,is subjected to degradation by diazotization to form a substancefragment having a free terminal aldehyde group. The substance fragmentis then reacted through its aldehyde group with the amino group of thesubstrate to form a Schiff's base, which is then converted (viareduction) to a secondary amine. The advantage of end-point attachmentof heparin, especially reducing end point attachment (as described abovein EP0086186B1), is that the biological activity of the heparin moietyis maximized due to enhanced availability of the antithrombininteraction sites as compared with attachment elsewhere in heparinmoiety.

The anticoagulant entity may be covalently attached to the outermostlayer of cationic polymer via a linker. Thus, in one embodiment, theanticoagulant entity is covalently attached via a linker.

In one embodiment, the linker comprises a secondary amine. Arepresentative procedure for covalently bonding a heparin moiety to apolymer via a secondary amine is described in EP0086186B1.

In one embodiment, the linker comprises a secondary amide. Arepresentative procedure for covalently bonding a heparin moiety to apolymer via an amidation reaction involving N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) or1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is set out inWO2012/123384A1 (incorporated herein by reference in its entirety).

In one embodiment, the linker comprises a 1,2,3-triazole. Arepresentative procedure for covalently bonding a heparin moiety to apolymer via a 1,2,3-triazole linkage is described in WO2010/029189A2(Carmeda AB, incorporated herein by reference in its entirety). Thedocument describes the azide- or alkyne-functionalization of apolyimine; the preparation of alkyne- and azide-functionalized heparin(both native and nitrous acid degraded heparin); and reactions to linkthe derivatised heparin to the derivatised polymer via a 1,2,3-triazolelinker.

In one embodiment, the linker comprises a thioether. A representativeprocedure for covalently bonding a heparin moiety to a polymer via athioether linkage is described in WO2011/110684A1 (Carmeda A B et al.,incorporated herein by reference in its entirety).

Cationic Polymer

The cationic polymer may be a straight chain polymer but is more usuallya branched chain polymer such as a hyperbranched polymer. In oneembodiment the cationic polymer is a branched cationic polymer. Thecationic polymer is optionally cross-linked. In one embodiment, thecationic polymer comprises primary/secondary amine groups. In oneembodiment, the cationic polymer is a polyamine, which is optionallycross-linked. The cationic polymer (e.g. polyamine), suitably hasmolecular weight of 5 kDa-3,000 kDa, such as 5 kDa-2,000 kDa, 5kDa-1,500 kDa, 5 kDa-1,000 kDa, 5 kDa-800 kDa, 5 kDa-500 kDa, 5 kDa-300kDa or 5 kDa-200 kDa or 800 kDa-3,000 kDa. When the cationic polymer(e.g. polyamine) is cross-linked, it is suitably cross-linked using analdehyde cross-linker such as crotonaldehyde and/or glutaraldehyde. Inone embodiment, the cationic polymer is a polyalkyleneimine e.g.polyethyleneimine.

The cationic polymer forms part of a layer-by-layer coating of cationicpolymer and anionic polymer, which is formed by alternately treating thesurface of the solid object with layers of cationic and anionic polymer.A bilayer is defined herein as one layer of cationic polymer and anionicpolymer. In the layer-by-layer coating, the cationic polymer istypically applied before the anionic polymer i.e. a surface of the solidobject is typically first treated with a first layer of cationic polymer(step i) in claim 1), upon which a first layer of anionic polymer isapplied (step ii) in claim 1). Depending on the number of bilayersrequired, further layers of cationic polymer and anionic polymer may beapplied (step iii) in claim 1). When the final (which may be also thefirst) bilayer of cationic and anionic polymer is completed, a layer ofcationic polymer is then applied (corresponding to step iv) in claim 1).This layer (i.e. the outermost layer) of cationic polymer is thentreated with an anticoagulant entity, so as to covalently attach theanticoagulant entity to the layer of cationic polymer. Thus, the outercoating layer of cationic polymer can be said to “comprise” ananticoagulant entity. In the layer-by-layer coating, the innermost layeris a layer of cationic polymer and the outermost layer is an outercoating layer of cationic polymer to which the anticoagulant entity iscovalently attached (see FIG. 1).

In one embodiment, the cationic polymer of step i) is a polyamine, whichis optionally cross-linked. In one embodiment, the cationic polymer ofstep iv) is a polyamine, which is optionally cross-linked. In oneembodiment, the cationic polymer of step i) is the same as the cationicpolymer of step iv).

WO2012/123384A1 (Gore Enterprise Holdings, Inc. et al., incorporatedherein by reference in its entirety) discloses a device with a coatingcomprising a plurality of hyperbranched polymer molecules bearinganticoagulant entities, in particular heparin. Such hyperbranchedpolymer molecules may be utilised in the outermost layer of cationicpolymer i.e. such hyperbranched polymers may be used as the cationicpolymer of step iv), and then modified to bear anticoagulant entities instep v).

Anionic Polymer

Anionic polymers suitable for the invention carry deprotonatedfunctional groups from the groups consisting of —COOH, —SO₃H and —PO₃H₂.Thus, in one embodiment, the anionic polymer is a polymer comprisinggroups selected from —CO₂, —SO₃ ⁻, —PO₃H⁻ and —PO₃ ²⁻. Suitably, theanionic polymer is a polymer comprising —SO₃ ⁻ groups. More suitably,the deprotonated functional groups carried by the anionic polymerconsist of —SO₃ ⁻ groups.

The anionic polymer is suitably an anionic glycosaminoglycan orpolysaccharide. The anionic characteristics of the polymer typicallyderive from carboxylate or sulfate groups along the polymer chain. Thus,in one embodiment, the anionic polymer is a glycosaminoglycan orpolysaccharide bearing carboxylate and/or sulfate groups, in particulara glycosaminoglycan bearing carboxylate and/or sulfate groups. Theanionic polymer may be branched or unbranched. In one embodiment, theanionic polymer and the anticoagulant entity are not the same.

In one embodiment, the anionic polymer is optionally cross-linked.

In one embodiment, the anionic polymer is selected from the groupconsisting of dextran sulfate, hyaluronic acid,poly(2-acrylamido-2-methyl-1-propanesulfonic acid),poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile)acrylonitrile, poly(acrylic acid), polyanetholesulfonic acid,poly(sodium 4-styrenesulfonate), poly(4-styrenesulfonic acid-co-maleicacid), poly(vinyl sulfate), polyvinylsulfonic acid and salts thereof.Suitably, the anionic polymer is dextran sulfate.

Dextran sulfate is a sulfated polymer of anhydroglucose. The degree ofsulfation and consequently the sulfur content of the dextran sulfate canvary.

In some embodiments the sulfur content is between 10% and 25% by weight,e.g. the sulfur content is between 15% and 20% by weight.

In one embodiment, the anionic polymer is characterized by having atotal molecular weight of 750 kDa-10,000 kDa, such as 1,000 kDa-10,000kDa. In one embodiment, the anionic polymer is characterized by having atotal molecular weight of 650 kDa-1,000 kDa, e.g. 750 kDa-1,000 kDa. Inone embodiment, the anionic polymer is characterized by having a totalmolecular weight of 1,000 kDa-4,500 kDa e.g. 2,000 kDa-4,500 kDa. In oneembodiment, the anionic polymer is characterized by having a totalmolecular weight of 4,500 kDa-7,000 kDa. In one embodiment, the anionicpolymer is characterized by having a total molecular weight of 7,000kDa-10,000 kDa. Suitably, the total molecular weight of the anionicpolymer is measured according to Evaluation Method G.

In one embodiment, the anionic polymer is characterized by having asolution charge density of between >4 μeq/g and 7 μeq/g, such asbetween >5 μeq/g and 7 μeq/g. Suitably, the solution charge density ofthe anionic polymer is measured according to Evaluation Method H.

Coating Bilayer(s) of Cationic and Anionic Polymer

The process of the invention involves forming a solid object having asurface comprising a layered coating of cationic and anionic polymer. Asexplained above, a bilayer is defined herein as one layer of cationicand anionic polymer (see FIG. 1).

The layered coating comprises one or more coating bilayers, e.g. 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more or 10 or more coating bilayers. When more than one coatingbilayer is applied, steps i) and ii) are repeated i.e. step iii) is notoptional. In one embodiment of the process of the invention, step iii)is not optional. In this embodiment, step iii) is repeated as many timesas is necessary to achieve the required number of coating bilayers e.g.1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or9 times. In one embodiment of the process of the invention, in stepiii), steps i) and ii) are repeated between 1 and 10 times, such 1, 2,3, 4, 5 or 6 times.

When step iii) is not optional (i.e. when steps i) and ii) are repeatedone or more times) the precise process conditions of each repeat neednot be identical (e.g. the salt type and/or concentration used intreating the surface with an anionic polymer in step ii) need not beidentical in each repetition). In an embodiment, the process conditions(e.g. the salt type and/or concentration used in treating the surfacewith an anionic polymer in step ii)) are identical in each repetition.

Process Steps

The present invention provides a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer comprises ananticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a solution charge density of >4μeq/g;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

It should be noted that steps i)-v) are carried out sequentially in thegiven order i.e. each of steps i)-iv) is implicitly followed by “andthen”. This does not preclude one or more additional steps being carriedout between each of specified steps i)-v). Thus, in one embodiment, theprocess of the invention additionally comprises a step between step i)and step ii), between step ii) and step iii), between step iii) and stepiv) or between step iv) and step v).

It should be understood, for example, that washing steps may beperformed between the specified process steps.

The present inventors have found that, surprisingly, the saltconcentration of step ii) (i.e. the salt concentration present when theanionic polymer coating layer(s) is (are) applied) impacts the resultingcharacteristics of the coating of the solid object, in particular thethromboresistant properties of the final solid object. The presentinventors have found that when step ii) is carried out at a saltconcentration of 0.25 M-5.0 M, the resulting characteristics of thecoating of the solid object, in particular the thromboresistantproperties of the final solid object can be improved, as shown inExamples 2a and 3a.

In one embodiment, step ii) is carried out at a salt concentration of0.25 M-4.0 M, such as 0.25 M-3.0 M, 0.5 M-3.0 M, 1.0 M-3.0 M, 1.5 M-3.0M, 0.25 M-1.5 M, 0.5 M-1.5 M, 0.75 M-1.5 M or 1.0 M-2.0 M, inparticular, at a salt concentration of 1.0 M-3.0 M, such as 1.0 M-2.0 Mor 0.75 M-1.5 M or 1.5 M-3.0 M.

In one embodiment, the salt is an inorganic salt. Suitably, the salt isselected from the group consisting of a sodium salt, a potassium salt, amagnesium salt, a calcium salt, a lithium salt, an ammonium salt, abarium salt and a strontium salt.

In one embodiment, the salt is an inorganic sodium salt.

In one embodiment, the salt is selected from the group consisting ofsodium chloride, sodium sulfate, sodium hydrogen phosphate and sodiumphosphate.

In one embodiment, the salt is sodium chloride.

In one embodiment, the salt is not sodium chloride.

In one embodiment, the salt is sodium chloride at a concentration of0.25 M-3.0 M, e.g. 0.5 M-3.0 M, e.g. 1.0 M-3.0 M, e.g. 1.5 M-3.0 M.

In one embodiment, the salt is sodium sulfate at a concentration of 0.25M-1.5 M, e.g. 0.5 M-1.5 M, e.g. 0.75 M-1.5 M.

In one embodiment, the salt is sodium hydrogen phosphate at aconcentration of 0.25 M-3.0 M, e.g. 0.5 M-3.0 M, e.g. 1.0 M-3.0 M, e.g.1.0 M-2.0 M.

In one embodiment, the salt is sodium phosphate at a concentration of0.25 M-3.0 M e.g. 0.5 M-3.0 M e.g. 1.0 M-3.0 M. e.g. 1.0 M-2.0 M.

Prior to step i) (treating the surface of the solid object with acationic polymer) the surface of the solid object can optionally besubjected to a pre-treatment step.

The pre-treatment step may be a cleaning step to improve adhesion andsurface coverage of the subsequent coating. Suitable cleaning agentsinclude solvents such as alcohols, solutions with high pH like solutionscomprising a mixture of an alcohol and an aqueous solution of ahydroxide compound (e.g. sodium hydroxide), sodium hydroxide solution assuch, solutions containing tetramethyl ammonium hydroxide (TMAH), acidicsolutions like Piranha (a mixture of sulfuric acid and hydrogenperoxide), basic Piranha solution, and other oxidizing agents includingcombinations of sulfuric acid and potassium permanganate or differenttypes of peroxysulfuric acid or peroxydisulfuric acid solutions (also asammonium, sodium, and potassium salts), or by subjecting the solidobject to plasma in air, argon or nitrogen atmosphere or combinationsthereof.

Thus, in one embodiment, the process of the invention additionallycomprises a pre-treatment step before step i). Suitably, thepre-treatment step is a cleaning step.

Alternatively, a pre-treatment step may involve overlaying the surfaceof the solid object to be coated according to steps i)-v) with amaterial such as a polymer or primer coating layer, prior to theapplication of steps i)-v). This “preparative” coating layer could, forexample, allow the surface of the solid object to be coated to be“sculpted” or modified to create a desired surface topography or texturein order to optimize the subsequent layered coating process. Theadditional coating layer could also improve the adherence of thesubsequent layered coating, in particular helping to maintain itsintegrity during processing. An example of such a priming coating layeron a solid object is a coating layer applied using chemical vapourdeposition (CVD). Another example of such a priming coating layer on asolid object is a coating of polydopamine or an analogue thereof.

In one embodiment, the pre-treatment step comprises treating a surfaceof the solid object with a polymer selected from the group consisting ofa polyolefin, polyisobutylene, ethylene-α-olefin copolymers, an acrylicpolymer, an acrylic copolymer, polyvinyl chloride, polyvinyl methylether, polyvinylidene fluoride, polyvinylidene chloride, a fluoropolymer(e.g. expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene(PTFE), fluorinated ethylene-propylene (FEP), perfluorocarboncopolymers, e.g. tetrafluoroethylene perfluoroalkylvinyl ether(TFE/PAVE) copolymers, copolymers of tetrafluoroethylene (TFE) andperfluoromethyl vinyl ether (PMVE), copolymers of TFE with functionalmonomers that comprise acetate, alcohol, amine, amide, sulfonate,functional groups and the like as described in U.S. Pat. No. 8,658,707(W.L. Gore and Associates, incorporated herein by reference in itsentirety, as well as combinations thereof), polyacrylonitrile, apolyvinyl ketone, polystyrene, polyvinyl acetate, an ethylene-methylmethacrylate copolymer, an acrylonitrile-styrene copolymer, an ABSresin, Nylon 12, a block copolymer of Nylon 12, polycaprolactone, apolyoxymethylene, a polyether, an epoxy resin, a polyurethane,rayon-triacetate, cellulose, cellulose acetate, cellulose butyrate,cellophane, cellulose nitrate, cellulose propionate, a cellulose ether,carboxymethyl cellulose, a chitin, polylactic acid, polyglycolic acid, apolylactic acid-polyethylene oxide copolymer, polyethylene glycol,polypropylene glycol, polyvinyl alcohol, an elastomeric polymer such assilicone (e.g. polysiloxane or a substituted polysiloxane), apolyurethane, a thermoplastic elastomer, an ethylene vinyl acetatecopolymer, a polyolefin elastomer, an EPDM rubber, and mixtures thereof.

In one embodiment, is provided a process for the manufacture of a solidobject as described herein, consisting of steps i)-v) as defined hereini.e. the solid object has no additional coating layers beyond thoseresulting from steps i)-v).

A solid object coated according to the process of the invention may besterilized. Suitable sterilization processes include, but are notlimited to, sterilization using ethylene oxide, vapour hydrogenperoxide, plasma phase hydrogen peroxide, dry heat, autoclave steamsterilization, chlorine dioxide sterilization, gamma ray sterilizationor electron beam sterilization.

As shown in Example 7, solid objects coated according to the process ofthe invention were subjected to increased temperature and humidity andretained their thromboresistant properties. Conditions of increasedtemperature and humidity can act as a mimic for the rigorous conditionsof sterilization, in particular ethylene oxide sterilization. Hence, asolid object coated according to the process of the invention isexpected to be stable to sterilization.

Coating Properties

Typically, the coating layer will have an average total thickness ofabout 10 nm to about 1000 nm, e.g. about 10 nm to about 800 nm, e.g.about 10 mM to about 500 nm, about 10 nm to about 400 nm, about 10 nm toabout 300 nm, about 10 nm to about 200 nm or about 10 nm to about 100nm. Coating thickness can be measured using a suitable coating thicknessanalyser or gauge, by using X-ray photoelectron spectroscopy with depthprofiling (see Evaluation Method J) or by using Quartz CrystalMicrobalance with Dissipation (see Evaluation Method 0). Suitably, thecoating thickness is measured using Evaluation Method 0.

In one embodiment, the solid object coated according to the process ofthe invention has anticoagulant entity activity (in particular heparinactivity) of at least 1 μmol/cm² of surface e.g. at least 2 μmol/cm² ofsurface, at least 3 μmol/cm² of surface, at least 4 μmol/cm² of surface,or at least 5 μmol/cm² of surface for binding of ATIII, suitablymeasured according to Evaluation Method B.

In one embodiment, a thromboresistant surface of the solid object hasanticoagulant entity activity (in particular heparin activity) of atleast 1 μmol/cm² of surface e.g. at least 2 μmol/cm² of surface, atleast 3 μmol/cm² of surface, at least 4 μmol/cm² of surface, or at least5 μmol/cm² of surface for binding of ATIII, suitably measured accordingto Evaluation Method B.

In one embodiment, the solid object coated according to the process ofthe invention has anticoagulant entity activity (in particular heparinactivity) of at least 5 μmol/cm² of surface e.g. at least 12 μmol/cm² ofsurface, at least 20 μmol/cm² of surface, at least 50 μmol/cm² ofsurface for binding of HCII, suitably measured according to EvaluationMethod M.

In one embodiment, a thromboresistant surface of the solid object hasanticoagulant entity activity (in particular heparin activity) of atleast 5 μmol/cm² of surface e.g. at least 12 μmol/cm² of surface, atleast 20 μmol/cm² of surface, at least 50 μmol/cm² of surface forbinding of HCII, suitably measured according to Evaluation Method M.

In one embodiment, the solid object coated according to the process ofthe invention has blood contact performance of at least 80% preservedplatelets, e.g. at least 85% preserved platelets, e.g. at least 90%preserved platelets, suitably measured according to Evaluation Method E.

In one embodiment, a thromboresistant surface of a solid object hasblood contact performance of at least 80% preserved platelets, e.g. atleast 85% preserved platelets, e.g. at least 90% preserved platelets,suitably measured according to Evaluation Method E.

In one embodiment, the solid object coated according to the process ofthe invention has an F1+2 value of <10,000 μmol/L e.g. less than 7,500μmol/L, less than 5,000 μmol/L or less than 4,000 μmol/L, suitablymeasured according to Evaluation Method F.

In one embodiment, a thromboresistant surface of a solid object has anF1+2 value of <10,000 μmol/L, less than 7,500 μmol/L, less than 5,000μmol/L or less than 4,000 μmol/L, suitably measured according toEvaluation Method F.

In one embodiment, the anticoagulant entity is a heparin moiety, whereinthe solid object has heparin concentration of at least 1 μg/cm², e.g. atleast 2 μg/cm², at least 4 μg/cm², at least 5 μg/cm², or at least 6μg/cm², suitably measured according to Evaluation Method A.

Zeta potential profiles of the solid objects coated according to theprocess of the invention may suitably be measured using EvaluationMethod D. A typical zeta potential profile of a solid object coatedaccording to the process of the invention is shown in FIG. 22 whichindicates the various parameters that can be used qualify and define azeta potential profile, in particular the isoelectric point (IEP) (1A)which corresponds to a particular pH value at which the zeta potentialis 0 mV; the global minimum of the curve corresponding to the pH (2A) atwhich the zeta potential (2B) is at a minimum; and the delta value (A)which corresponds to the difference between the zeta potential at theglobal minimum (3A) and the zeta potential at pH 9 (3B).

Similar zeta (ζ) potential profiles have been obtained for solid objectscoated according to the process of the invention with dextran sulfates4-7 as can be seen in FIGS. 7-13 (Examples 4a and 4b). Thus, the zetapotential profile can be viewed as a potential fingerprint for solidobjects coated according to the process of the invention, at least inits preferred aspects. According to this potential fingerprint,preferably the IEP (1A) is below pH 3, the global minimum of the curve(2A) is below pH 5 and the delta value i.e. the difference between thezeta potential at the global minimum (3A) and the zeta potential at pH 9(3B), is at least 20 mV. These parameters are suitably measuredaccording to Evaluation Method D.

Solid objects coated according to the process of the invention suitablyhave a zeta potential profile with an isoelectric point (IEP) of belowpH 3 because the acidic nature of the heparin dominates on the coatedsurface. By contrast, inert polymer materials will have an IEP atapproximately pH 4. The swelling properties of samples having acidicgroups is seen towards the alkaline region. The swelling will force theshear plane in the direction of the bulk and lower absolute zetapotential values (closer to 0 mV) should be obtained. A high delta valueof the zeta potential correlates with high thromboresistant propertiese.g. when evaluated according to Evaluation Methods B, M, E or F.Furthermore, higher salt concentration used in the process of theinvention gives lower absolute zeta potential values in the alkalineregions irrespective of the salt tested, yet again correlating with theantithrombin binding values. Without being limited by theory, this canpotentially be explained with an increased access of antithrombin toheparin molecules in a coating that can undergo swelling.

Therapeutic Methods

Solid objects, in particular medical devices coated according to theprocess of the invention as described hereinabove are of use in medicaltherapy.

In one aspect of the invention is provided a solid object (in particulara medical device) coated according to the process of the inventiondescribed hereinabove for use in treating tissue in the human or animalbody. The tissue to be treated includes any body cavity, space, orhollow organ passage(s) such as blood vessels, the urinary tract, theintestinal tract, nasal cavity, neural sheath, intervertebral regions,bone cavities, oesophagus, intrauterine spaces, pancreatic and bileducts, rectum, and those previously intervened body spaces that haveimplanted vascular grafts, stents, prosthesis, or other type of medicalimplants. In yet another aspect of the invention, a solid object (e.g.medical device) coated according to a process of the invention asdescribed hereinabove may be deployed to treat aneurysms in the brain.

The coated solid object (in particular medical device) as describedherein can be of use in the removal of obstructions such as emboli andthrombi from blood vessels, as a dilation device to restore patency toan occluded body passage, as an occlusion device to selectively delivera means to obstruct or fill a passage or space, and as a centeringmechanism for transluminal instruments like catheters.

In one embodiment is provided a solid object (in particular a medicaldevice such as a stent, graft or stent-graft) coated according to theprocess of the invention as described hereinabove for use in theprevention or treatment of stenosis or restenosis in a blood vessel ofthe human body. In another embodiment is provided a solid object (inparticular a medical device such as a stent, graft or stent-graft)coated according to the process of the invention as describedhereinabove for use in the prevention or treatment of stenosis orrestenosis in a blood vessel of the human body, where previously placedeluting constructs have failed. In another embodiment, a solid object(in particular a medical device such as a stent, graft or stent-graft)coated according to the process of the invention as describedhereinabove can be used to establish or maintain arteriovenous accesssites, e.g. those used during kidney dialysis. In a further embodiment,a solid object (in particular a medical device such as a stent, graft orstent-graft e.g. a vascular graft) coated according to the process ofthe invention described hereinabove may be used to redirect flow aroundan area of blockage or vessel narrowing. In another embodiment, a solidobject (in particular a medical device such as a stent, graft orstent-graft) coated according to the process of the invention asdescribed hereinabove may be deployed to restore patency to an area ofdiseased vessel or to exclude an aneurysm. In yet another embodiment, asold object (in particular a medical device such as a stent, graft orstent-graft) coated according to the process of the invention asdescribed hereinabove may be deployed to reinforce a diseased vesselfollowing angioplasty. In yet another embodiment, a solid object (inparticular a medical device such as a stent, graft or stent-graft)coated according to the process of the invention as describedhereinabove may be deployed in the brain using balloon assisted or coilassisted procedures.

In one embodiment, a solid object (in particular a medical device)coated according to the process of the invention as describedhereinabove can be used for Percutaneous Transluminal Angioplasty (PTA)in patients with obstructive disease of the peripheral arteries.

In another aspect of the invention is provided a method for theprevention or treatment of stenosis or restenosis which comprisesimplanting into said blood vessel in the human body a solid object (inparticular a medical device) coated according to the process of theinvention as described hereinabove.

Further Embodiments of the Invention

Embodiments and preferences described above with respect to the processof the invention apply equally to embodiments below.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisingcationic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

wherein,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a solution charge density of >4μeq/g;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer;

and wherein the anionic polymer is characterized by having (a) a totalmolecular weight of 650 kDa-10,000 kDa; and (b) a solution chargedensity of >4 μeq/g.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisinganionic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

wherein,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a solution charge density of >4μeq/g;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising anionic polymer; and wherein theanionic polymer is characterized by having (a) a total molecular weightof 650 kDa-10,000 kDa; and (b) a solution charge density of >4 μeq/g.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer comprises ananticoagulant entity, consisting of the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a solution charge density of >4μeq/g;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 650kDa-10,000 kDa; and (b) a solution charge density of >4 μeq/g. Suitablythe anionic polymer is applied to the surface at a salt concentration of0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 650kDa-1,000 kDa; and (b) a solution charge density of >4 μeq/g. Suitablythe anionic polymer is applied to the surface at a salt concentration of0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 1,000kDa-4,500 kDa; and (b) a solution charge density of >4 μeq/g. Suitablythe anionic polymer is applied to the surface at a salt concentration of0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 4,500kDa-7,000 kDa; and (b) a solution charge density of >4 μeq/g. Suitablythe anionic polymer is applied to the surface at a salt concentration of0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 7,000kDa-10,000 kDa; and (b) a solution charge density of >4 μeq/g. Suitablythe anionic polymer is applied to the surface at a salt concentration of0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer comprises ananticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is a polymer comprising —SO₃ ⁻ groups,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a sulfur content between 10% and25% by weight of the anionic polymer;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer comprises ananticoagulant entity, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of cationic polymer with ananticoagulant entity, thereby to covalently attach the anticoagulantentity to the outermost layer of cationic polymer;

wherein,

the anionic polymer is characterized by having a total molecular weightof 650 kDa-10,000 kDa;

the anionic polymer is dextran sulfate;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer to which iscovalently bound an anticoagulant entity; the anionic polymer is apolymer comprising —SO₃ ⁻ groups and wherein the anionic polymer ischaracterized by having (a) a total molecular weight of 650 kDa-10,000kDa; and (b) a sulfur content between 10% and 25% by weight of theanionic polymer.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisingcationic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

wherein,

the anionic polymer is a polymer comprising —SO₃ ⁻ groups,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a sulfur content between 10% and25% by weight of the anionic polymer;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisingcationic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

wherein,

the anionic polymer is characterized by having a total molecular weightof 650 kDa-10,000 kDa; the anionic polymer is dextran sulfate;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising cationic polymer; the anionicpolymer is a polymer comprising —SO₃ ⁻ groups and wherein the anionicpolymer is characterized by having (a) a total molecular weight of 650kDa-10,000 kDa; and (b) a sulfur content between 10% and 25% by weightof the anionic polymer.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisinganionic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

wherein,

the anionic polymer is a polymer comprising —SO₃ ⁻ groups,

the anionic polymer is characterized by having (a) a total molecularweight of 650 kDa-10,000 kDa; and (b) a sulfur content between 10% and25% by weight of the anionic polymer;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a process for the manufacture of a solidobject having a surface comprising a layered coating of cationic andanionic polymer wherein the outer coating layer is a layer comprisinganionic polymer, comprising the steps of:

i) treating a surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) optionally repeating steps i) and ii) one or more times;

wherein,

the anionic polymer is characterized by having a total molecular weightof 650 kDa-10,000 kDa;

the anionic polymer is dextran sulfate;

and wherein,

step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

In one embodiment is provided a solid object having a surface comprisinga layered coating of cationic and anionic polymer, wherein the outercoating layer is a layer comprising anionic polymer; the anionic polymeris a polymer comprising —SO₃ ⁻ groups and wherein the anionic polymer ischaracterized by having (a) a total molecular weight of 650 kDa-10,000kDa; and (b) a sulfur content between 10% and 25% by weight of theanionic polymer.

Clauses of the Invention

Additional Clauses of the Invention:

-   1. A process for the manufacture of a solid object having a surface    comprising a layered coating of cationic and anionic polymer wherein    the outer coating layer comprises an anticoagulant entity,    comprising the steps of:    -   i) treating a surface of the solid object with a cationic        polymer;    -   ii) treating the surface with an anionic polymer;    -   iii) optionally repeating steps i) and ii) one or more times;    -   iv) treating the surface with a cationic polymer; and    -   v) treating the outermost layer of cationic polymer with an        anticoagulant entity, thereby to covalently attach the        anticoagulant entity to the outermost layer of cationic polymer;    -   wherein,    -   the anionic polymer is characterized by having (a) a total        molecular weight of 650 kDa-10,000 kDa; and (b) a solution        charge density of >4 μeq/g;    -   and wherein,    -   step ii) is carried out at a salt concentration of 0.25 M-5.0 M.-   2. A process for the manufacture of a solid object according to    clause 1, wherein the anionic polymer is dextran sulfate.-   3. A process for the manufacture of a solid object according to    clause 1 or clause 2, wherein the anionic polymer is characterized    by having a total molecular weight of 750 kDa-10,000 kDa, such as    1,000 kDa-10,000 kDa.-   4. A process for the manufacture of a solid object according to any    one of clauses 1 to 3, wherein the anionic polymer is characterized    by having a solution charge density of between >4 μeq/g and 7 μeq/g,    such as between >5 μeq/g and 7 μeq/g.-   5. A process for the manufacture of a solid object according to any    one of clauses 1 to 4, wherein step ii) is carried out at a salt    concentration of 0.25 M-4.0 M, such as 0.25 M-3.0 M.-   6. A process for the manufacture of a solid object according to any    one of clauses 1 to 5, wherein the salt is selected from the group    consisting of sodium chloride, sodium sulfate, sodium hydrogen    phosphate and sodium phosphate, and in particular is sodium    chloride.-   7. A process for the manufacture of a solid object according to any    one of clauses 1 to 6, wherein the cationic polymer of step i) is a    polyamine, which is optionally cross-linked; and/or wherein the    cationic polymer of step iv) is a polyamine, which is optionally    cross-linked.-   8. A process for the manufacture of a solid object according to any    one of clauses 1 to 7, wherein the anticoagulant entity is a heparin    moiety e.g. an end-point attached heparin moiety which is connected    through its reducing end.-   9. A process for the manufacture of a solid object according to any    one of clauses 1 to 8, wherein the solid object is a    thromboresistant medical device.-   10. A solid object having a surface comprising a layered coating of    cationic and anionic polymer, wherein the outer coating layer is a    layer comprising cationic polymer to which is covalently bound an    anticoagulant entity;    -   and wherein the anionic polymer is characterized by having (a) a        total molecular weight of 650 kDa-10,000 kDa; and (b) a solution        charge density of >4 μeq/g.-   11. A solid object according to clause 10, wherein the anionic    polymer is characterized by having a total molecular weight of 650    kDa-1,000 kDa, or 1,000 kDa-4,500 kDa or 4,500 kDa-7,000 kDa or    7,000 kDa-10,000 kDa.-   12. A solid object according to any one of clauses 10 or 11, wherein    the anionic polymer is applied to the surface at a salt    concentration of 0.25 M-5.0 M, such as 0.25 M-4.0 M or 0.25 M-3.0 M.-   13. A process for the manufacture of a solid object having a surface    comprising a layered coating of cationic and anionic polymer wherein    the outer coating layer is a layer comprising cationic polymer,    comprising the steps of:    -   i) treating a surface of the solid object with a cationic        polymer;    -   ii) treating the surface with an anionic polymer;    -   iii) optionally repeating steps i) and ii) one or more times;        and    -   iv) treating the surface with a cationic polymer;    -   wherein,    -   the anionic polymer is characterized by having (a) a total        molecular weight of 650 kDa-10,000 kDa; and (b) a solution        charge density of >4 μeq/g;    -   and wherein,    -   step ii) is carried out at a salt concentration of 0.25 M-5.0 M.-   14. A solid object having a surface comprising a layered coating of    cationic and anionic polymer, wherein the outer coating layer is a    layer comprising cationic polymer; and wherein the anionic polymer    is characterized by having (a) a total molecular weight of 650    kDa-10,000 kDa; and (b) a solution charge density of >4 μeq/g.-   15. A process for the manufacture of a solid object having a surface    comprising a layered coating of cationic and anionic polymer wherein    the outer coating layer is a layer comprising anionic polymer,    comprising the steps of:    -   i) treating a surface of the solid object with a cationic        polymer;    -   ii) treating the surface with an anionic polymer;    -   iii) optionally repeating steps i) and ii) one or more times;    -   wherein,    -   the anionic polymer is characterized by having (a) a total        molecular weight of 650 kDa 10,000 kDa; and (b) a solution        charge density of >4 μeq/g;    -   and wherein,    -   step ii) is carried out at a salt concentration of 0.25 M-5.0 M.

Advantages

Solid objects coated according to the process of the invention, at leastin some embodiments, are expected to have one or more of the followingmerits or advantages:

-   -   A coating of the anticoagulant entity having uniform        distribution and being comparatively smooth can be obtained e.g.        as determined using Evaluation Method C (toluidine blue staining        test) or Evaluation Method I (SEM);    -   A uniform coating may be obtained which will mask the intrinsic        properties of the solid object, for example to improve the        thromboresistant properties of a device irrespective of the        material of its manufacture;    -   A coating with good anticoagulant entity activity such as        heparin activity can be obtained e.g. as determined using        Evaluation Method B or M;    -   A thromboresistant coating which does not leach anticoagulant        entity e.g. heparin, due to its covalent attachment and        therefore has a long lifetime may be obtained;    -   A coating whose properties are preserved upon sterilization        (e.g. with EO) may be obtained;    -   A self-healing coating may be obtained due to the possibility of        reversible forming of ionic interactions between the layers;    -   A coating with good biocompatibility can be obtained e.g. as        determined by using Evaluation Method N;    -   A coating which may reduce the need for systemic administration        of anticoagulant e.g. heparin, and reduce the likelihood of        contact activation e.g. as determined using Evaluation Method E        (platelets) and/or Evaluation Method F (blood loop) may be        obtained;    -   A solid object having a combination of anti-inflammatory        properties as determined by using Evaluation Method N and        thromboresistance can be obtained which may be beneficial in        certain applications e.g. cardiovascular applications;    -   An analytical or separation device with good binding capacity to        biomolecules may be obtained; and    -   An analytical or separation device with long heparin activity        life time may be obtained.

The invention embraces all combinations of indicated groups andembodiments of groups recited above.

Abbreviations

Ac acetylABS acrylonitrile butadiene styreneATIII antithrombin IIICNS central nervous systemCPB cardiopulmonary bypassCVC central venous catheterCVD chemical vapour depositionDa daltonDI deionizedEDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimideEO ethylene oxideEPDM ethylene propylene diene monomer (M-class)ePTFE expanded polytetrafluoroethyleneFEP fluorinated ethylene-propyleneGPC gel permeation chromatographyHCII heparin cofactor IIHIT heparin induced thrombocytopeniaIEP isoelectric pointM molar concentrationMBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloridePAVE perfluoroalkylvinyl etherPES-Na sodium polyethylene sulfatePTA percutaneous transluminal angioplastyPIC peripherally inserted central catheterPMVE perfluoromethyl vinyl etherPTFE polytetrafluoroethylenePUR polyurethanePVC polyvinyl chlorideRGD arginylglycylaspartic acidSEM scanning electron microscopy/microscopeSPDP N-succinimidyl 3-(2-pyridyldithio)propionateTFE tetrafluoroethyleneTMAH tetramethyl ammonium hydroxideTMB 3,3′,5,5′-tetramethylbenzidineVA ventriculoatrialVP ventriculoperitonealXPS X-ray photoelectron spectroscopy

EXAMPLES

General Procedures

Chemicals

Isopropanol, sodium dihydrogen phosphate dihydrate, sodium sulfate andsodium chloride are available from Sigma Aldrich and VWR Chemicals andmay be used as received. Heparin of pharmacopea quality was treated withnitrous acid, essentially as described in EP0086186A1 and used in theExamples. Polyamines are available from vendors as described in U.S.Pat. No. 9,101,696B2. Dextran sulfates were purchased from variousvendors as indicated in Table 1 of Example 1. Deionized (DI) water wasused in the Examples below.

Materials

PVC tubing was purchased from Flex Tubing Products. PUR tubing waspurchased from NewAge Industries. Stainless steel coupons were purchasedfrom Helab Mekano AB.

Evaluation Methods

The parameter being evaluated by each method is given in parentheses.

Evaluation Method A: Heparin Concentration Test (Quantitative HeparinAttachment)

Quantification of surface immobilized heparin can be performed bycomplete degradation of heparin followed by colorimetric determinationof the reaction products released into solution. Degradation is achievedby reacting the heparin surface with an excess of sodium nitrite underacidic conditions. The degradation products, mainly disaccharides, arequantified colorimetrically in a reaction with MBTH(3-methyl-2-benzothiazolinone hydrazone hydrochloride), essentially asdescribed in Smith R. L. and Gilkerson E (1979), Anal Biochem 98,478-480, which is incorporated herein by reference in its entirety.

Evaluation Method B: Heparin Activity Test (Quantitative HeparinFunction Using ATIII)

For solid objects coated according to the process of the inventioncomprising a heparin coating, the heparin activity of the device can bemeasured by measuring the ability, or capacity, of the heparin to bindantithrombin III (ATIII) as described by Pasche, et al. in “A binding ofantithrombin to immobilized heparin under varying flow conditions”(Artif. Organs 1991; 15:281-491, incorporated herein by reference in itsentirety) and Larsen M. L, et al. in “Assay of plasma heparin usingthrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA” (S-2238)(Thromb. Res. 1978; 13:285-288, incorporated herein by reference in itsentirety), and can be used to evaluate a solid object's thromboresistantproperties. Washed samples are incubated with an excess of antithrombinin solution to saturate all available antithrombin-binding sites of theheparin surface. Non-specifically adsorbed antithrombin is rinsed awayusing a salt solution. Subsequently, antithrombin specifically bound tothe surface bound heparin is released by incubating with a solution ofheparin at high concentration. Finally, the antithrombin released fromthe heparin surface is measured in a thrombin inhibition assay, based ona chromogenic thrombin substrate. The results are expressed as picomolesantithrombin III (ATIII) bound per apparent square centimeter of device(μmol ATIII/cm² solid object surface). The apparent solid object surfacearea does not take into account multiple covered surfaces nor porosityconsiderations of a solid object composed of a porous material. If thesurface of the solid object is porous, the effect of porosity on surfacearea is not considered for these calculations. For example, the apparentsurface area of a cylindrical tubular ePTFE vascular graft (which ismade of a porous material) with heparin immobilized on substratematerial comprising the inner surface of the tubular graft is calculatedas it is for any cylindrical geometry as 2ττrL: where r is the graftinner radius; L is the axial length; and π is the number pi. This methodcan be used to measure the activity of any anticoagulant entity withATIII binding activity.

Evaluation Method C: Toluidine Blue Staining Test (Heparin Distribution)

Heparin distribution is evaluated using toluidine blue stainingsolution. The solution is prepared by dissolving 200 mg of toluidineblue in 1 L of water. The samples are subjected to the staining solutionfor 2 minutes prior to extensive water rinse. A blue/violet stainingindicates that negatively charged heparin molecules are homogenouslydistributed in the outer coating layer.

Evaluation Method D: Zeta Potential Measurement (Indicator of SurfaceCharge)

Zeta potential, as an indicator of surface charge, of the coating isdetermined on a SurPASS instrument. The measurement is conducted bycirculating an electrolyte over the surface, which is by standard a 1 mMsolution of a simple electrolyte such as KCl or NaCl. The resultingstreaming potential is measured and used to determine the zetapotential. The zeta potential of coating is determined in the pH range 3to 9 via addition of acid or base to the solution respectively. The zetapotential is calculated using Eq 1 below, as described in T. Luxbacher,The ZETA guide, Principles of the streaming potential technique, firstedition, published by Anton Paar GMBH, ISBN 978-3-200-03553-9(incorporated herein by reference in its entirety).

$\begin{matrix}{\zeta = {\frac{dU}{dp} \times \frac{\eta}{ɛ \times ɛ_{0}} \times K_{B}}} & \left( {{Eq}\mspace{14mu} 1} \right)\end{matrix}$

dU/dP=slope of streaming potential vs differential pressure,

K_(B)=electrolyte conductivity

η=electrolyte viscosity

ε=dielectric coefficient of electrolyte

ε₀=vacuum permittivity.

Evaluation Method E: Blood Loop Evaluation Test (Measurement of PlateletLoss)

Blood contact evaluation can be performed on a coated object to evaluateits thromboresistant properties. A procedure which may be used when thesolid object is a tubular device such as a piece of PVC tubing is asfollows. Firstly, the luminal side of the coated tubing is washed with0.15 M saline solution for 15 hours at a flow of 1 mL/min to ensurecomplete wetting and removal of any loosely bound anticoagulant entity,such that a stable surface remains. The washed tubing is then incubatedin a Chandler loop model performed essentially according to Andersson etal. (Andersson, J.; Sanchez, J.; Ekdahl, K. N.; Elgue, G.; Nilsson, B.;Larsson, R. J Biomed Mater Res A 2003, 67(2), 458-466, incorporatedherein by reference in its entirety) at 20 rpm. The platelets from freshblood and from the blood collected from the loops are counted in a cellcounter to measure the loss of platelets. A great loss of plateletsindicates poor thromboresistant performance of the surface. Conversely aminimal loss of platelets indicates a thromboresistant surface.

Evaluation Method F: Blood Loop Evaluation Test (for Measurement ofF1+2)

The determination of F1+2 (prothrombin fragment) is used as anactivation marker for coagulation (i.e. as an indirect measurement ofthrombin). F1+2 is directly proportional to the formation of thrombinand interpreted as an indirect measurement of thrombin generation, andcan be used to evaluate a solid object's thromboresistant properties.Quantitative determination of F1+2 in plasma is performed with anenzymatic immunoanalysis, by using a standard ELISA kit (Enzyme-LinkedImmuno Sorbent Assay) (Enzygnost F1+2 ELISA, OPBDG03, Siemens). The F1+2antigen in the sample couples to the antibodies entrapped on the coatedsurface of 96-well microtiter plate and subsequently detected by aperoxidase conjugated anti-F1+2 antibody. The amount of coupledperoxidase is measured by addition of a specific substrate,3,3′,5,5′-tetramethylbenzidine (TMB). The enzymatic conversion of thesubstrate to chromogen is stopped by addition of diluted sulfuric acid.Absorbance at 450 nm in the wells is proportional to the concentrationof F1+2 in the sample. The concentration of the samples is determined bycomparison to a standard curve with known concentrations of F1+2.

Evaluation Method G: Molecular Weight of Anionic Polymers Such asDextran Sulfate in Solution

Determination of the molecular weight of a dextran sulfate sample isperformed on a gel permeation chromatography (GPC) instrument. Thedextran sulfate samples are dissolved in a water-based elution media andanalyzed on a GPC instrument suitable for the molecular weight range1,000 Da-100,000 Da (superose column) or 100,000 Da-2,000,000 Da(sephacryl column). A dextran sulfate standard of an appropriatemolecular weight is used to verify the accuracy of the calibrationcurve. Polymers such as dextran sulfate are disperse molecules i.e. havea distribution of molecular weights, which can be described withdifferent molecular weight averages. The commonly reported value is theweight average molecular weight (Mw). See Odian G., Principles ofPolymerization, Third edition, Section 1.4 Molecular weight, p. 24(incorporated herein by reference in its entirety) which explains thetheory on determination of molecular weights of polymers using GPCtechniques. The molecular weight of anionic polymers other than dextransulfate can also be determined using this method.

Evaluation Method H: Solution Charge Density of Anionic Polymers Such asDextran Sulfate in Solution

Quantitative determination of charge density is performed on a MütekParticle Charge Detector via titration of polyelectrolyte solutions(0.001 M) (polydiallyldimethylammonium chloride (Poly-Dadmac) and sodiumpolyethylene sulfate (PES-Na)). Samples are dissolved in water (maximumviscosity allowed 6000 mPas) to a concentration of 0.06 g/L. The pH isadjusted to 3 for all sample solutions. 10 mL per sample solution isadded each measurement followed by titration of appropriatepolyelectrolyte solution at an interval of 1 unit per 3 seconds. See S.Farris et al., Charge Density Quantification of PolyelectrolytePolysaccharides by Conductometric Titration: An Analytical ChemistryExperiment, J. Chem. Educ., 2012, 89 (1), pp 121-124 (incorporatedherein by reference in its entirety). The solution charge density ofanionic polymers other than dextran sulfate can also be determined usingthis method.

Evaluation Method I: Scanning Electron Microscopy with Energy DispersiveX-Ray Spectroscopy (Coating Coverage and Uniformity)

TM3000 is a table-scanning electron microscope (SEM) manufactured byHitachi that is used to obtain information about e.g. a samplethickness, topography (surface structure) and surface coverage. A highermagnification can be achieved with table SEM compared to traditionallight microscopes as it is electrons used to create the image. TheTM3000 is also equipped with Quantax70. This is an Energy DispersiveX-ray Spectrometer (EDS) used to determine the chemical composition ofthe sample. In addition, there is a rotation/tilt table as accessory tofacilitate analysis of different parts of the sample. The sample ismounted on a holder with carbon tape (also acts as grounding) and thenplaced in the test chamber. The chamber is evacuated to a lower pressurebefore evaluation of the sample can commence. SEM technology is based onthe scanning of an electron beam across the sample, some of theelectrons being reflected backscattered electrons, while others executesecondary electrons. A detector is used to measure the current generatedby the reflected backscattered electrons. The current is imaged on adisplay where each pixel corresponds to the position of the sample. Abright pixel is obtained if many electrons are reflected (high electrondensity) and a darker pixel is obtained if few electrons (low electrondensity) are reflected.

Evaluation Method J: X-Ray Photoelectron Spectroscopy with DepthProfiling (XPS) (Coating

Thickness) X-ray Photoelectron Spectroscopy (XPS or ESCA) is the mostwidely used surface characterization technique providing non-destructivechemical analysis of solid materials. Samples are irradiated withmono-energetic X-rays causing photoelectrons to be emitted from the top1 nm-10 nm of the sample surface. An electron energy analyzer determinesthe binding energy of the photoelectrons. Qualitative and quantitativeanalysis of all elements except hydrogen and helium is possible, atdetection limits of ˜0.1-0.2 atomic percent. Analysis spot sizes rangefrom 10 μm to 1.4 mm. It is also possible to generate surface images offeatures using elemental and chemical state mapping. Depth profiling ispossible using angle-dependent measurements to obtain non-destructiveanalyses within the top 10 nm of a surface, or throughout the coatingdepth using destructive analysis such as ion etching.

Evaluation Method K: Increased Temperature and Humidity Test (GeneralModel for Sterilization Stability)

The solid object coated according to the process of the invention isplaced in a breathable polyethylene pouch (e.g. a Tyvek pouch). Thepouch is placed in a climate chamber (e.g. Climacell) at 40° C. and 50%relative humidity for 1 week followed by 2 hours drying in a vacuumchamber. After performing this general model for sterilizationstability, the thromboresistant properties/activation of the coatedobject is assessed e.g. using Evaluation Method E or F.

Evaluation Method L: Stability to Ethylene Oxide

The solid object coated according to the process of the invention isplaced in a breathable polyethylene pouch (e.g. a Tyvek pouch) andsubjected to at least 12 hours preconditioning at 50° C. and 60%relative humidity followed by 2 hours exposure of ethylene oxide at apressure of 366 mBar and 50° C. The chamber is then degassed at 50° C.for at least 10 hours. Sterilization by ethylene oxide may be performedat Synergy Health Ireland Ltd. After sterilization, the thromboresistantproperties/activation of the coated object is assessed e.g. usingEvaluation Method E or F.

Evaluation Method M: Heparin Activity Test (Quantitative HeparinFunction Using HCII)

For solid objects coated according to the process of the inventioncomprising a heparin coating, the heparin activity of the device can bemeasured by measuring the ability, or capacity, of the heparin to bindheparin cofactor II (HCII) as described in WO2009/064372A2 (GoreEnterprise Holdings, Inc.; incorporated herein by reference in itsentirety) by measuring the ability, or capacity, of the heparin to binda known quantity of heparin cofactor 11 (HCII), using an assay asdescribed by Larsen M. L., et al., in “Assay of plasma heparin usingthrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238).”Thromb Res 13:285-288 (1978) and Pasche B., et al., in “A binding ofantithrombin to immobilized heparin under varying flow conditions.”Artif. Organs 1991; 15:281-491), and can be used to evaluate a solidobject's thromboresistant properties. The results are expressed aspicomoles heparin cofactor II (HCII) bound per apparent squarecentimetre of solid object surface (μmol HCll/cm² solid object surface).The apparent solid object surface area does not take into accountmultiple covered surfaces nor porosity considerations of a devicecomposed of a porous material. If the surface of the device is porous,the effect of porosity on surface area is not considered for thesecalculations. For example, the apparent surface area of a cylindricaltubular ePTFE vascular graft (which is made of a porous material) withheparin immobilized on substrate material comprising the inner surfaceof the tubular graft is calculated as it is for any cylindrical geometryas 2ττrL: where r is the graft inner radius; L is the axial length; andπ is the number pi. This method can be used to measure the activity ofany anticoagulant entity with HCII binding activity.

Evaluation Method N—Surface Biocompatibility

The biocompatibility of a surface of a solid object coated according toa process of the invention can be assessed as described in Lappegard, K.T 2008, J. Biomed. Mater. Res. Vol 87, 129-135 (incorporated herein byreference in its entirety). A procedure which may be used to evaluatethe inflammatory response is as follows. Firstly, the coated solidobject is washed with 0.15 M saline solution for 15 min. The wettedcoated solid object is placed in heparinized PVC tubing containing wholeblood and left to rotate in a circulating loop at 20 rpm (see Ekdahl K.N., Advances in Experimental Medicine and Biology, 2013, 735, 257-270(incorporated herein by reference in its entirety) for a representativeprocedure). After incubation, the blood is centrifuged for 15 min, 3220g at 4° C. The plasma is frozen in aliquots at −70° C. for lateranalysis of cytokines. Plasma samples are analyzed using multiplexcytokine assay (Bio-Plex Human Cytokine 27-Plex Panel, Bio-RadLaboratories, Hercules, Calif.) according to the method described byLappegard et al. (above).

The negative control is an empty loop of heparinized PVC without anydevice. This represents a non-inflammatory control for which theincubated blood should demonstrate no or minimal amount of inflammatorymarkers. The positive control is an empty loop of non-heparinized PVCwithout any device. This represents an inflammatory control for which agreater amount of inflammatory markers should be observed. The controlsare included for ensuring the quality of the experiment and the blood.

Evaluation Method O—Quartz Crystal Microbalance with Dissipation(Coating Thickness)

Q-sense E4 is a crystal microbalance with dissipation (QCM-D) monitoringinstrument. QCM-D is a technique for measurement of both mass andstructural properties of molecular layers and may be seen as anultrasensitive weighing deceive.

A QCM sensor consists of a thin quartz disc where AT-cut crystals arethe most commonly used. The quartz disc is placed between two electrodesand by applying a voltage to the quartz crystal it can be made tooscillate at its resonance frequency. Changes in mass on the quartzsurface induces a change in frequency of the oscillating crystal relatedthrough the Sauerbrey relationship (see Rodahl, M., et al., Quartzcrystal microbalance setup for frequency and Q factor measurements ingaseous and liquid environments. Review of scientific environments,1995. 66(7): p.3924-3930. (incorporated herein by reference in itsentirety). Coating thickness of solid objects coated according to theprocess of the invention are reported as dry coating thickness.

Evaluation Method P—Molecular Weight Determination of the HeparinFragment Fractions

The molecular weight of Heparin fragment fractions are determined byanalytical gel permeation chromatography (GPC) on a system consisting oftwo Superdex columns in series (S-75 and S-200) essentially according toUSP<209>Low Molecular Weight Heparin Molecular Weight Determinations.Peak positions are identified based on the elution profile of the 2ndInternational Standard for Low Molecular Weight Heparin for MolecularWeight Calibration (NIBSC, UK), where the least retarded peak of thestandard is a disaccharide.

Evaluation Method Q—Heparin Fragment Concentration Determination

The quantities of isolated heparin fragment in solution are estimated byanalyzing the uronic acid content by the carbazole assay (Bitter, T.;Muir, H. M., Anal. Biochem.,1962, (4), 330-334), related to a heparinstandard curve.

Example 1: Processes for Coating a Solid Object (Layered Coating ofCationic and Anionic Polymer, with Outer Coating Layer of AnticoagulantEntity)

General Coating Process—Tubing

The luminal surface of a section of tubing (e.g. PVC or PUR tubing) iscoated with a layer-by-layer coating of cationic polymer and anionicpolymer using essentially the method described by Larm et al. inEP0086186A1, EP0495820B1 and EP0086187A1 (all incorporated herein byreference in their entirety).

Specifically, the luminal surface of the tubing is firstly cleaned withisopropanol and an oxidizing agent. The coating bilayers are built-up byalternating adsorption of a cationic polymer (polyamine, 0.05 g/L inwater) and an anionic polymer (dextran sulfate, 0.1 g/L in water). Thepolyamine is crosslinked with a difunctional aldehyde (crotonaldehyde).The dextran sulfate raw material is varied as specified in each of theExamples below, and applied in the presence of various sodium salts atvaried concentrations, again as specified in each Example below. Everypair of polyamine and sulfated polysaccharide is called one bilayer i.e.a bilayer is defined as one layer of cationic and anionic polymer andthe same conditions are used for building up of each bilayer. Theluminal surface of the tubing is coated with three bilayers (see FIG. 1for a solid object coated with a single bilayer). A final, outermostlayer of polyamine is then adsorbed.

Heparin is then immobilized to the outermost layer of polyamine viareductive amination, essentially as described by Larm et al. inEP0086186A1 and EP0495820B1 (both incorporated herein by reference intheir entirety).

General Coating Process—Steel Coupons

Any solid object can be coated using the general coating processdescribed above for tubing. In the Examples below where a steel couponwas utilized, the entire surface of the coupon was coated.

Dextran Sulfates Used in Examples 1.1-1.56

The evaluated dextran sulfates were purchased from different vendors aspresented in Table 1.

TABLE 1 Dextran sulfates evaluated in the Examples Solution chargedensity** Dextran sulfate No. Vendor Mw* [kDa] [μeq/g] (pH3) 1 SigmaAldrich 50 6.1 (Reference example dextran sulfate) 2 Tdb Consultancy 1005.4 (Reference example dextran sulfate) 3 Tdb Consultancy 600 3.0(Reference example dextran sulfate) 4 Tdb Consultancy 800 6.3 5 pKChemicals A/S 4000 6.2 6 Alfa Aesar 5000 5.3 7 Sigma Aldrich 8000 6.4*Weight average molecular weight (Mw) determined according to EvaluationMethod G **Solution charge density determined according to EvaluationMethod H

Example 1.1: Preparation of Coating on PVC Tubing Using Dextran Sulfate1 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 1, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.2: Preparation of Coating on PVC Tubing Using Dextran Sulfate1 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 1, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.3: Preparation of Coating on PVC Tubing Using Dextran Sulfate2 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 2, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.4: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 0.05 M.

Example 1.5: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 0.1 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 0.1 M.

Example 1.6: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.7: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 1.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 1.0 M.

Example 1.8: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.9: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 2.6 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 2.6 M.

Example 1.10: Preparation of Coating on PVC Tubing Using Dextran Sulfate3 and NaCl Concentration of 3.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 3, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.11: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 0.05 M.

Example 1.12: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 0.1 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 0.1 M.

Example 1.13: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.14: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 1.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 1.0 M.

Example 1.15: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.16: Preparation of Coating on PVC Tubing Using Dextran Sulfate4 and NaCl Concentration of 3.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 4, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.17: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 0.05 M

PVC tubing was coated according to the general procedure describedabove. Dextran sulfate 5, see Table 1, was applied at NaCl concentrationof 0.05 M.

Example 1.18: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.19: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 0.5 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 0.5 M.

Example 1.20: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 0.85 M.

Example 1.21: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 1.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 1.0 M.

Example 1.22: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.23: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and NaCl Concentration of 3.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.24: Preparation of Coating on PVC Tubing Using Dextran Sulfate6 and NaCl Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 6, see Table 1, was applied at NaClconcentration of 0.05 M

Example 1.25: Preparation of Coating on PVC Tubing Using Dextran Sulfate6 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 6, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.26: Preparation of Coating on PVC Tubing Using Dextran Sulfate6 and NaCl Concentration of 0.5 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 6, see Table 1, was applied at NaClconcentration of 0.5 M.

Example 1.27: Preparation of Coating on PVC Tubing Using Dextran Sulfate6 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 6, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.28: Preparation of Coating on PVC Tubing Using Dextran Sulfate6 and NaCl Concentration of 3.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 6, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.29: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.05 M.

Example 1.30: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.1 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.1 M.

Example 1.31: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.32: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.5 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.5 M.

Example 1.33: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.85 M.

Example 1.34: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 1.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 1.0 M.

Example 1.35: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.36: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 2.6 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 2.6 M.

Example 1.37: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 3.0 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.38: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and NaCl Concentration of 3.4 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 3.4 M.

Example 1.39: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂HPO₄ Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂HPO₄concentration of 0.05 M.

Example 1.40: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂HPO₄ Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂HPO₄concentration of 0.25 M.

Example 1.41: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂HPO₄ Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂HPO₄concentration of 0.85 M.

Example 1.42: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂HPO₄ Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂HPO₄concentration of 1.7 M.

Example 1.43: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂SO₄ Concentration of 0.05 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂SO₄concentration of 0.05 M.

Example 1.44: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂SO₄ Concentration of 0.25 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂SO₄concentration of 0.25 M.

Example 1.45: Preparation of Coating on PVC Tubing Using Dextran Sulfate5 and Na₂SO₄ Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 5, see Table 1, was applied at Na₂SO₄concentration of 0.85 M.

Example 1.46: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and Na₂HPO₄ Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at Na₂HPO₄concentration of 0.85 M.

Example 1.47: Preparation of Coating on PVC Tubing Using Dextran Sulfate7 and Na₂SO₄ Concentration of 0.85 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at Na₂SO₄concentration of 0.85 M.

Example 1.48: Preparation of Coating on PUR Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.05 M

PUR tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.05 M.

Example 1.49: Preparation of Coating on PUR Tubing Using Dextran Sulfate7 and NaCl Concentration of 0.25 M

PUR tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 0.25 M.

Example 1.50: Preparation of Coating on PUR Tubing Using Dextran Sulfate7 and NaCl Concentration of 1.7 M

PUR tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 1.51: Preparation of Coating on PUR Tubing Using Dextran Sulfate7 and NaCl Concentration of 3.0 M

PUR tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 7, see Table 1, was applied at NaClconcentration of 3.0 M.

Example 1.52: Preparation of Coating on Steel Coupon Using DextranSulfate 7 and NaCl Concentration of 0.05 M

A steel coupon (15.0 mm×3.35 mm) was coated according to the generalprocedure described above. Dextran sulfate 7, see Table 1, was appliedat NaCl concentration of 0.05 M.

Example 1.53: Preparation of Coating on Steel Coupon Using DextranSulfate 7 and NaCl Concentration of 0.25 M

A steel coupon (15.0 mm×3.35 mm) was coated according to the generalprocedure described above. Dextran sulfate 7, see Table 1, was appliedat NaCl concentration of 0.25 M.

Example 1.54: Preparation of Coating on Steel Coupon Using DextranSulfate 7 and NaCl Concentration of 1.7 M

A steel coupon (15.0 mm×3.35 mm) was coated according to the generalprocedure described above. Dextran sulfate 7, see Table 1, was appliedat NaCl concentration of 1.7 M.

Example 1.55: Preparation of Coating on Steel Coupon Using DextranSulfate 7 and NaCl Concentration of 3.0 M

A steel coupon (15.0 mm×3.35 mm) was coated according to the generalprocedure described above. Dextran sulfate 7, see Table 1, was appliedat NaCl concentration of 3.0 M.

Example 1.56: Preparation of Coating on PVC Tubing Using Dextran Sulfate2 and NaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated according to the general proceduredescribed above. Dextran sulfate 2, see Table 1, was applied at NaClconcentration of 1.7 M.

Example 2a: Normalized Heparin Activity of Coated PVC Tubing UsingDifferent Dextran Sulfates at Varying NaCl Concentration

Heparin activity of PVC tubing coated according to Examples 1.11-1.19,1.21-1.32, 1.34-1.38 (corresponding to dextran sulfates 4, 5, 6 and 7)at varying NaCl concentrations was measured as set out in EvaluationMethod B (Heparin activity test).

All coated solid objects tested exhibited heparin activity of at least 1μmol/cm² when determined by Evaluation Method B. Heparin activity valuesshown in Table 2 below are normalized to the highest heparin activityvalue observed for coated PVC tubing with dextran sulfate 5 at 1.7 MNaCl (Example 1.22).

TABLE 2 Normalized heparin activity (%) of coated PVC tubing withdextran sulfates 4, 5, 6 and 7 at varying NaCl concentration Dextransulfate No. 4 5 6 7 Normalized heparin Example No. NaCl concentration[M] activity [%] 1.11/1.17/1.24/1.29 0.05 28 46 27 42 1.12/—/—/1.30 0.1033 — — 62 1.13/1.18/1.25/1.31 0.25 46 62 48 60 —/1.19/1.26/1.32 0.50 5938 78 1.14/1.21/—/1.34 1.00 93 95 — 82 1.15/1.22/1.27/1.35 1.70 92 100 62 98 —/—/—/1.36 2.60 — — — 86 1.16/1.23/1.28/1.37 3.00 71 99 50 64—/—/—/1.38 3.40 — — — 38

Normalized heparin activity values for PVC tubing coated with dextransulfates 4, 5, 6 and 7 at 0.25 M and 1.7 M NaCl are shown in FIG. 2. Itcan be seen from FIG. 2 that although both salt concentrations led tocoatings with acceptable thromboresistant properties, the use of thehigher salt concentration (1.7 M) in the step of adding the dextransulfate layer led to higher heparin activity than use of the lower saltconcentration (0.25 M). It can be seen from Table 2 that the use of saltconcentrations of less than 0.25 M resulted in lower heparin activities.The highest heparin activities were obtained using the dextran sulfateswith charge density above 6 μeq/g (dextran sulfates 4, 5, 7).

Example 2b: Normalized Heparin Activity of Coated PVC Tubing UsingDextran Sulfate 5, with Different Salts at Varied Concentration

Heparin activity of PVC tubing coated according to Examples 1.17, 1.18,1.20, 1.22, and 1.39-1.45 (corresponding to dextran sulfate 5) usingNaCl, Na₂HPO₄ or Na₂SO₄, at varying concentrations was measured as setout in Evaluation Method B (Heparin activity test).

All coated solid objects tested exhibited heparin activity of at least 1μmol/cm². Heparin activity values shown in Table 3 below are normalizedto the highest heparin activity observed for coated PVC tubing withdextran sulfate 5 at 1.70 M NaCl (Example 1.22)

TABLE 3 Normalized heparin activity (%) of coated PVC tubing (dextransulfate 5) using different salts at varied concentration Dextran sulfateNo. 5 Salt Na₂HPO₄ Na₂SO₄ NaCl Example No. Salt concentration [M]Normalized heparin activity [%] 1.39/1.43/1.17 0.05 7 34 461.40/1.44/1.18 0.25 11 18 62 1.41/1.45/1.20 0.85 25 30 32 1.42/—/1.221.70 36 * 100 * Not soluble in water at 1.7M

Normalized heparin activity values from Table 3 are shown in FIG. 3. Itcan be seen from FIG. 3 that the beneficial effect on heparin activityof using a higher salt concentration in the step of adding the dextransulfate layer is shown by a range of salts. The highest heparin activityvalues were obtained using sodium chloride.

Example 2c: Normalized Heparin Activity of Coated PVC Tubing UsingDextran Sulfates 5 and 7, with Different Salts at 0.85 M Concentration

Heparin activity of PVC tubing coated according to Examples 1.20, 1.33,1.41 and 1.45-1.47 (corresponding to dextran sulfate 5 and 7 using NaCl,Na₂HPO₄ or Na₂SO₄ at 0.85 M was measured as set out in Evaluation MethodB (Heparin activity test).

All coated solid objects tested exhibited heparin activity of at least 1μmol/cm². Heparin activity values shown in Table 4 below are normalizedto the highest heparin activity value observed for Example 1.22.

TABLE 4 Normalized heparin activity (%) of coated PVC tubing (dextransulfate 5 and 7) using different salts at 0.85M concentration SaltDextran concentration Example No. sulfate no. [M] Na₂HPO₄ Na₂SO₄ NaCl1.41/1.45/1.20 5 0.85 25 30 32 1.46/1.47/1.33 7 0.85 43 38 45

It can be seen that the use of various salts, e.g. NaCl, Na₂HPO₄ andNa₂SO₄, does not significantly affect the heparin activity values. Thesalt concentration will affect the heparin activity regardless of thesalt used.

Example 2d: Normalized Heparin Activity of Various Coated Solid ObjectsUsing Dextran Sulfate 7, with NaCl at Varied Concentration

Heparin activity of various coated solid objects according to Examples1.29, 1.31, 1.35, 1.37, and 1.48-1.55 (corresponding to dextran sulfate7) using NaCl at varied concentration was measured as set out inEvaluation Method B (Heparin activity test).

All coated solid objects tested exhibited heparin activity of at least 1μmol/cm². Heparin activity values shown in Table 5 below are normalizedto the highest heparin activity value observed for Example 1.22.

TABLE 5 Normalized heparin activity (%) of various coated solid objects(dextran sulfate 7) with NaCl at varied concentration Dextran Saltsulfate concentration Example No. No. [M] PVC PUR Steel 1.29/1.48/ 70.05 46 40 65 1.52 1.31/1.49/ 7 0.25 60 62 81 1.53 1.35/1.50/ 7 1.70 98110 135 1.54 1.37/1.51/ 7 3.00 64 72 88 1.55

It is evident from Table 5 that the salt concentration will affect theheparin activity regardless of the material of the solid object that hasbeen coated. Tubing made of polyurethane (PUR) and steel coupons werecoated with dextran sulfate 7 at varied salt concentrations and theresulting normalized heparin activity values show that there is a clearsalt dependence.

Example 2e: Normalized Heparin Activity of PVC Tubing Coated withFragments of Heparin (Octasaccharides) Using Dextran Sulfate 5 and aNaCl Concentration of 1.7 M

PVC tubing (I.D. 3 mm) was coated with fragments of Heparin (anoctasaccharide) according to the general procedure described above withDextran sulfate 5, see Table 1, applied at NaCl concentration of 1.7 M.

Heparin Fragment Fractions Prepared by Depolymerization of HeparinFollowed by Fractionation

Oligosaccharides, predominantly of the size of eight sugar units (octa),were prepared by partial nitrous acid cleavage of native heparinfollowed by fractionation by gel chromatography. An octasaccharideproduced by nitrous cleavage is the shortest fragment that can contain afunctional active sequence (Thunberg L. et al, FEBS Letters 117 (1980),203-206).

Depolymerization of heparin: 10 g of heparin sodium was dissolved in 36ml of water by stirring overnight. 0.30 g NaNO₂ was added to the heparinsolution and allowed to dissolve. The solution was acidified to pH 2.5by addition of 4M HCl. After a total reaction time of 2 h at roomtemperature, the solution was neutralized by addition of 4M NaOH.

The degradation mixture was separated based on molecular size by gelpermeation chromatography (GPC), where portions of 3 ml were applied tothe column (HiLoad 26/600 Superdex 30 pg, mobile phase 0.15 M NaCl) at aflow rate of 2.5 ml/min. The collected fractions (3 ml) were analyzedfor aldehyde by the MBTH reaction, essentially as described in Smith R.L. and Gilkerson E (1979), Anal Biochem 98, 478-480. A broad peakcentred on the elution position of the octasaccharide was collected. Thecombined oligosaccharide elution fractions from several preparative runswere concentrated by evaporation to a volume of 18 ml andre-chromatographed on the same column. For all re-chromatographic runsthree fractions, representing deca-, octa- and hexasaccharide fragments,were collected and pooled.

The collected fractions were analysed by Evaluation Method P. The “hexa”fraction consists of a major peak representing hexasaccharide and ashoulder representing octasaccharide. The “octa” fraction consists of amajor peak representing octasaccharide with a shoulder representinghexasaccharide and a minor shoulder representing decasaccharide. The“deca” fraction consists of a major peak representing decasaccharidewith a shoulder representing octasaccharide and a minor shoulderrepresenting dodecasaccharide.

The concentration of the heparin fragment fractions was determined byEvaluation Method Q (see table below).

“Hexa” “Octa” “Deca” concentration mg/ml 4.0 6.2 3.6

Immobilisation of Octasaccharide

The PVC tubing was coated with sixteen ml of the “octa” fraction dilutedwith 84 ml of 0.05 M NaCl, the octa fraction was then immobilized to theoutermost layer of polyamine via reductive amination, essentially asdescribed by Larm et al. in EP0086186A1 and EP0495820B1 (bothincorporated herein by reference in their entirety).

Evaluation by Toluidine Staining of PVC Tubing Coated with HeparinFragments

The oligosaccharide coated surface was subjected to a toluidine bluestaining test as set out in Evaluation Method C. An intense blue/violetcolor was observed on the luminal surface of the tubing indicating anextensive covalent attachment of the heparin fragments. The homogenousstaining obtained for tested tubing indicates formation of a uniformcoating.

Evaluation of Heparin Density of PVC Tubing Coated with HeparinFragments

The heparin density of the surface was determined by Evaluation Method Aand the results are shown in the table below.

Evaluation of Heparin Activity of PVC Tubing Coated with HeparinFragments

The heparin activity of the octasaccharide coated surface (Example 2e)was determined by Evaluation Method B. Heparin activity values shown inthe table below are normalized to the highest heparin activity observedfor coated PVC tubing with dextran sulfate 5 at 1.70 M NaCl (Example1.22).

Heparin density Heparin activity Example no. (μg/cm²) (%) 2e 5.6 5 1.226.5 100

Although the heparin density value of the octasaccharide coating(Example 2e) and the heparin coating (Example 1.22) were similar, the ATbinding capacity (heparin activity; ‘HA’) of the octasaccharide coatingswas low compared to the heparin coating. However, this is to be expectedconsidering the relatively low anti-FXa activity exhibited by theoctasaccharide fraction in solution (data not shown). Thus, theoctasaccharide fragments appear to substantially retain their AT-bindingcapacity after immobilization.

Example 3a: Heparin Concentration of Coated PVC Tubing Using DifferentDextran Sulfates at Varied NaCl Concentration

Heparin concentration of solid objects (PVC tubing) coated according toExamples 1.4-1.19, 1.21-1.32 and 1.34-1.38 (corresponding to dextransulfates 3, 4, 5, 6 and 7) at varying NaCl concentrations was measuredas set out in Evaluation Method A.

All coated solid objects tested exhibited heparin concentration of atleast 1 μg/cm². Heparin concentration values are shown in Table 6 below.

TABLE 6 Heparin concentration (μg/cm²) of coated PVC tubing with dextransulfates 3, 4, 5, 6 and 7 at varied NaCl concentration Dextran sulfateNo. 3 (Ref Salt concentra- Ex) 4 5 6 7 Example No. tion [M] Heparinconcentration [μg/cm²] 1.4/1.11/1.17/ 0.05 4.8 2.9 3.4 2.9 4.0 1.24/1.291.5/1.12/—/—/ 0.10 4.8 3.2 — — 4.7 1.30 1.6/1.13/1.18/ 0.25 5.1 3.0 3.73.9 5.7 1.25/1.31 —/—/1.19/1.26/1.32 0.50 — — 4.5 4.8 5.71.7/1.14/1.21/—/1.34 1.00 5.1 4.1 4.5 — 6.8 1.8/1.15/1.22/ 1.70 4.3 4.76.5 6.0 6.8 1.27/1.35 1.9/—/—/—/1.36 2.60 3.1 — — — 6.9 1.10/1.16/1.23/3.00 2.0 5.0 4.1 5.0 7.3 1.28/1.37 —/—/—/—/1.38 3.40 — — — — 6.2

Heparin concentration for PVC tubing coated with dextran sulfates 3, 4,5, 6 and 7 at 1.7 M NaCl are shown in FIG. 4. It can be seen from FIG. 4that there is a trend to higher heparin concentration from using adextran sulfate of higher molecular weight in the step of adding thedextran sulfate layer under these conditions. Dextran sulfate 3 is areference dextran sulfate in this example.

Heparin concentration for PVC tubing coated with dextran sulfates 3, 4,5, 6 and 7 at varied NaCl concentration are shown in FIG. 5. It can beseen from FIG. 5 that dextran sulfates 4, 5, 6 and 7 demonstrate a trendto higher heparin concentration with increased salt concentration (atleast up to 1.7 M) in the step of adding the dextran sulfate layer. Itcan be seen that the use of salt concentrations of less than 0.25 Mgenerally results in lower heparin activities. Dextran sulfate 3 doesnot follow this trend and when it is used the heparin concentrationlowers as the salt concentration in this step is increased. Dextransulfate 3 is a reference dextran sulfate in this example. Without beinglimited by theory, the inventors attribute this difference in trend tothe fact that dextran sulfate 3 has a much lower charge density thandextran sulfates 4, 5, 6 and 7.

Example 3b: Heparin Concentration of Coated PVC Tubing Using DextranSulfate 5, with Different Salts at Varied Concentration

Heparin concentration of PVC tubing coated according to Examples 1.17,1.18, 1.20, 1.22 and 1.39-1.45 (corresponding to dextran sulfate 5 usingNaCl, Na₂HPO₄ or Na₂SO₄, at varying concentrations was measured as setout in Evaluation Method A.

All coated solid objects tested exhibited heparin concentration of atleast 1 μg/cm². Heparin concentration values are shown in Table 7 below.

TABLE 7 Heparin concentration (μg/cm²) of coated PVC tubing (dextransulfate 5) using different salts at varied concentration Dextran sulfateNo. 5 5 5 Salt Na₂HPO₄ Na₂SO₄ NaCl Example No. Salt concentration [M]Heparin concentration [μg/cm²] 1.39/1.43/1.17 0.05 2.7 3.3 3.41.40/1.44/1.18 0.25 3.2 3.5 3.7 1.41/1.45/1.20 0.85 5.0 4.2 3.51.42/—/1.22 1.70 5.1 * 6.5 * Na₂SO₄ not soluble in water at 1.7M

Heparin concentration values from Table 7 are shown in FIG. 6. It can beseen from FIG. 6 that dextran sulfate 5 demonstrates a trend toincreased heparin concentration with increased salt concentration in thestep of adding the dextran sulfate layer, for a range of differentsalts.

Example 3c: Heparin Concentration of Coated PVC Tubing Using DextranSulfates 5 and 7, with Different Salts at 0.85 M Concentration

Heparin concentration of PVC tubing coated according to Examples 1.20,1.33, 1.41, 1.45, 1.46 and 1.47 (corresponding to dextran sulfate 5 and7 using NaCl, Na₂HPO₄ or Na₂SO₄ at 0.85 M was measured as set out inEvaluation Method A.

All coated solid objects tested exhibited heparin concentration of atleast 1 μg/cm². Heparin concentration values are shown in Table 8 below.

TABLE 8 Heparin concentration (μg/cm²) of coated PVC tubing (dextransulfates 5 and 7) using different salts at 0.85M concentration DextranSalt sulfate concentration Example No. No. [M] Na₂HPO₄ Na₂SO₄ NaCl1.41/1.45/1.20 5 0.85 5.0 4.2 3.5 1.46/1.47/1.33 7 0.85 3.3 3.6 3.7

It can be seen that the use of various salts, e.g. NaCl, Na₂HPO₄ andNa₂SO₄, does not significantly affect the heparin concentration values.

Example 3d: Heparin Concentration of Various Coated Solid Objects UsingDextran Sulfate 7, with NaCl at Varied Concentration

Heparin concentration of various solid objects according to Examples1.29, 1.31, 1.35, 1.37, and 1.48-1.55. (corresponding to dextran sulfate7 (12)) coated using NaCl at varied concentration was measured as setout in Evaluation Method A.

All coated solid objects tested exhibited heparin concentration of atleast 1 μg/cm². Heparin concentration values are shown in Table 9 below.

TABLE 9 Heparin concentration (μg/cm²) of various coated solid objects(dextran sulfate 7 with NaCl at varied concentration Dextran Saltsulfate concentration Example No. No. [M] PVC PUR Steel 1.29/1.48/ 70.05 4.0 2.9 7.2 1.52 1.31/1.49/ 7 0.25 5.7 2.5 7.7 1.53 1.35/1.50/ 71.70 6.8 3.5 10.4 1.54 1.37/1.51/ 7 3.00 7.3 4.1 8.9 1.55

It is evident from Table 9 that the salt concentration will affect theheparin concentration regardless of the material of the solid objectthat has been coated. Tubing made of polyurethane (PUR) and steelcoupons were coated with dextran sulfate 7 at varied salt concentrationsand the resulting heparin concentration values show that there is aclear salt dependence.

Example 4a: Zeta Potential Measurement of Coated PVC Tubing UsingDifferent Dextran Sulfates at 1.7 M and 0.25 M NaCl Concentration

The surface charge of PVC tubing coated according to Examples 1.1, 1.2,1.3, 1.6, 1.8, 1.13, 1.15, 1.18, 1.22, 1.25, 1.27, 1.31, 1.35 and 1.56(corresponding to dextran sulfates 1, 2, 3, 4, 5, 6 and 7) at variedNaCl concentration) was measured as set out in Evaluation Method D.

The zeta potential values for PVC coated tubing with dextran sulfates 1to 7 at 1.7 M NaCl are shown in Table 10.

TABLE 10 The zeta potential for PVC coated tubing with dextran sulfates1 to 7 at 1.7M NaCl Delta value pH (global Example No. Dextran sulfateNo. [mV] minimum) IEP 1.2 1 20 6.1 2.6 (Reference example dextransulfate) 1.56 2 18 5.6 2.6 (Reference example dextran sulfate) 1.8 3 134.7 2.1 (Reference example dextran sulfate) 1.15 4 30 4.4 2.6 1.22 5 284.5 2.5 1.27 6 41 3.8 2.3 1.35 7 40 4.2 2.4

Dextran sulfates 1 to 7 all have an IEP below pH 3. However, the lowermolecular weight dextran sulfates (Reference example dextran sulfates1-3) do not fulfill all the preferred features (i.e. the potentialfingerprint for solid objects coated according to the process of theinvention, described above). Dextran sulfates 1 and 2 have a globalminimum occurring at a pH higher than 5 and dextran sulfate 3 has adelta value which is lower than 20 mV. Solid objects of the inventioncoated with dextran sulfates 4 to 7 do fulfill these criteria.

The zeta potential for PVC coated tubing with dextran sulfates 3, 4 and5 at 1.7 M NaCl (corresponding to Examples 1.8, 1.15 and 1.22) are shownin FIG. 7.

The zeta potential for PVC coated tubing with dextran sulfates 3, 6 and7 at 1.7 M NaCl (corresponding to Examples 1.8, 1.27 and 1.35) are shownin FIG. 8.

The zeta potential values for PVC coated tubing with dextran sulfates 1to 7 at 0.25 M NaCl are shown in Table 11.

TABLE 11 The zeta potential for PVC coated tubing with dextran sulfates1 to 7 at 0.25M NaCl Delta value pH (global Example No. Dextran sulfateNo. [mV] minimum) IEP 1.1 1 17 4.6 2.6 (Reference example dextransulfate) 1.3 2 23 6.1 2.7 (Reference example dextran sulfate) 1.6 3 154.2 1.5 (Reference example dextran sulfate) 1.13 4 37 4.5 2.7 1.18 5 384.5 2.6 1.25 6 35 4.1 2.5 1.31 7 36 4.5 2.5

Dextran sulfates 1 to 7 all have an IEP below pH 3. However, the lowermolecular weight dextran sulfates (Reference example dextran sulfates1-3) do not fulfill all the preferred features (i.e. the potentialfingerprint for solid objects coated according to the process of theinvention, described above). Dextran sulfate 2 has a global minimumoccurring at a pH higher than 5 and dextran sulfates 1 and 3 have adelta value which is lower than 20 mV. Solid objects of the inventioncoated with dextran sulfates 4 to 7 do fulfill these criteria.

The zeta potential for PVC coated tubing with dextran sulfates 3, 4 and5 at 0.25 M NaCl (corresponding to Examples 1.6, 1.13 and 1.18) areshown in FIG. 9.

The zeta potential for PVC coated tubing with dextran sulfates 3, 6, and7 at 0.25 M NaCl (corresponding to Examples 1.6, 1.25 and 1.31) areshown in FIG. 10.

Example 4b: Zeta Potential Measurement of Coated PVC Tubing UsingDextran Sulfate 5 with Different Salts at Varied Concentration

The surface charge of PVC tubing coated according to Examples 1.18,1.22, 1.39-1.42 and 1.44-1.45 (all dextran sulfate 5) using NaCl,Na₂HPO₄ or Na₂SO₄, at varying concentrations was measured as set out inEvaluation Method D.

The zeta potential values for PVC coated tubing with dextran sulfate 5at different NaCl concentrations are shown in Table 12.

TABLE 12 The zeta potential for PVC coated tubing with dextran sulfate 5at different NaCl concentrations Salt pH concentration Delta value(global Example No. [M] [mV] minimum) IEP 1.18 0.25 38 4.5 2.4 1.22 1.728 4.5 2.5

The zeta potential profiles for PVC coated tubing with dextran sulfate 5at NaCl concentrations of 0.25 M and 1.7 M (corresponding to Examples1.18 and 1.22) are shown in FIG. 11 where the salt effect on the zetapotential is evident. The preferred features (i.e. the potentialfingerprint for solid objects coated according to the process of theinvention, described above) are fulfilled at 0.25 and 1.7 M NaClconcentration.

The zeta potential values for PVC coated tubing with dextran sulfate 5at different Na₂HPO₄ concentrations are shown in Table 13.

TABLE 13 The zeta potential for PVC coated tubing with dextran sulfate 5at different Na₂HPO₄ concentrations Salt pH concentration Delta value(global Example No. [M] [mV] minimum) IEP 1.39 0.05 20 4.2 2.6 1.40 0.2528 4.3 2.4 1.41 0.85 41 4.5 2.6 1.42 1.70 38 4.2 2.7

The zeta potential profiles for PVC coated tubing with dextran sulfate 5at Na₂HPO₄ concentrations of 0.25 M, 0.85 M and 1.7 M (corresponding toExamples 1.40, 1.41 and 1.42) are shown in FIG. 12 where it can be seenthat all the preferred features (i.e. the potential fingerprint forsolid objects coated according to the process of the invention,described above) are fulfilled using Na₂HPO₄ at differentconcentrations.

The zeta potential values for PVC coated tubing with dextran sulfate 5at different Na₂SO₄ concentrations are shown in Table 14.

TABLE 14 The zeta potential for PVC coated tubing with dextran sulfate 5at different Na₂SO₄ concentrations Salt pH concentration Delta value(global Example No. [M] [mV] minimum) IEP 1.44 0.25 40 4.5 2.6 1.45 0.8539 4.3 2.6 N/A** 1.7 * * * * Not soluble in water at 1.7M **N/A = Notapplicable

The zeta potential profiles for PVC coated tubing with dextran sulfate 5at Na₂SO₄ concentration of 0.25 M and 0.85 M (corresponding to Examples1.44 and 1.45) are shown in FIG. 13 where it can be seen that all thepreferred features (i.e. the potential fingerprint for solid objectscoated according to the process of the invention, described above) arefulfilled using Na₂SO₄ concentrations at different concentrations. It isevident from Tables 12, 13 and 14 that using different types of salt atdifferent concentrations will not significantly affect the zetapotential profile. It is also clear that there is a salt dependence forthe different salt types.

Example 5: Blood Contact Activation (Platelet Loss and F1+2) of CoatedPVC Tubing Using Different Dextran Sulfates at Varied NaCl Concentration

The percentage of platelets preserved and the F1+2 (prothrombinfragment) after blood exposure of PVC tubing coated according toExamples 1.1, 1.3, 1.13, 1.18, 1.25 and 1.31 (corresponding to dextransulfates 1, 2, 4, 5, 6 and 7) at varied NaCl concentration were measuredas set out in Evaluation Methods E and F, respectively.

The results are shown in Table 15 and FIGS. 14 and 15 (0.25 M NaClconcentration) and Table 16 and FIGS. 16 and 17 (1.7 M NaClconcentration).

As seen in the Tables and Figures, no significant platelet loss(platelet loss indicating thrombosis) was observed for solid objectscoated according to the process of the invention with dextran sulfates4, 5, 6 and 7 at 0.25 M and 1.7 M NaCl concentration. Thethromboresistant properties of the coatings were further confirmed bythe low F1+2 values (prothrombin fragment) observed for the same dextransulfates. The tubing coated with comparative dextran sulfate 1 withmolecular weight of 50 kDa and with comparative dextran sulfate 2 withmolecular weight of 100 kDa also showed significant thrombosis and highgeneration of prothrombin fragments compared with solid objects of theinvention coated with dextran sulfates 4-7.

The uncoated PVC tubing and the clotting example show significantthrombosis in this experiment.

TABLE 15 Preserved platelets (%) and F1 + 2 (pmol/L) of PVC tubingcoated with dextran sulfates 1, 2, 4, 5, 6 and 7 at 0.25M NaClconcentration N Preserved F1 + 2 (number Example No. Dextran sulfate No.platelets [%] [pmol/L] average) 1.1 1 53 15868 2 (Reference exampledextran sulfate) 1.3 2 0 186405 1 (Reference example dextran sulfate)1.13 4 93 741 1 1.18 5 96 791 1 1.25 6 99 854 2 1.31 7 97 1237 2Uncoated — 2 637658 — PVC example Clotting — 1 644465 — example

TABLE 16 Preserved platelets (%) and F1 + 2 (pmol/L) of PVC tubingcoated with dextran sulfates 1, 4, 5, 6 and 7 at 1.7M NaCl concentrationN Preserved F1 + 2 (number Example No. Dextran sulfate No. platelets [%][pmol/L] average) 1.2 1 27 248469 2 (Reference example dextran sulfate)1.15 4 97 2057 2 (Reference example dextran sulfate) 1.22 5 90 3609 31.27 6 92 2734 1 1.35 7 86 3112 1 Uncoated — 2 637658 — PVC exampleClotting — 1 644465 — example

Example 6: Toluidine Blue Staining of Coated PVC and PUR Tubing andSteel Coupons Using Different Dextran Sulfates at Varied SaltConcentration

PVC and PUR tubing and steel coupons coated according to Examples1.1-1.55 were subjected to a toluidine blue staining test as set out inEvaluation Method C.

A blue/violet color was observed on the luminal surface of the tubingand steel coupons indicating the covalent attachment of end-pointfunctionalized heparin. The homogenous staining obtained for testedsolid objects coated according to the process of the invention indicatesformation of a uniform coating (in particular uniform heparindistribution) which may be obtained using different dextran sulfates atsalt different concentrations, on different solid objects.

Example 7: Blood Contact Activation (Platelet Loss and F1+2) of CoatedPVC Tubing—Post Temperature and Humidity Test

PVC tubing coated according to Examples 1.13, 1.15, 1.22 and 1.35(corresponding to dextran sulfates 4, 5, and 7)) at varied NaClconcentration was exposed to increased temperature and relative humidity(40° C., 50% RH, 1 week, according to Evaluation Method K) prior toevaluation according to Evaluation Methods E (preserved platelets) and F(F1+2). The results are shown in Table 17 and FIGS. 18 and 19, and Table18 and FIGS. 20 and 21.

As seen in the Tables and Figures, for solid objects coated according tothe process of the invention using dextran sulfates 4, 5 and 7, there isno significant change in the preserved platelet and F1+2 values postexposure to increased temperature and humidity. Similar results wereobtained for solid objects coated according to the process of theinvention using dextran sulfates 4, 5 and 7 prepared at 0.25 M and 1.7 MNaCl concentration.

These results demonstrate that the thromboresistant properties of thecoated solid objects prepared according to the process of the inventionare retained in spite of exposure to rigorous conditions as increasedtemperature and humidity.

TABLE 17 Preserved platelets (%) and F1 + 2 (pmol/L) of PVC tubingcoated with dextran sulfate 4 at 0.25M NaCl concentration - before andafter exposure to increased temperature and humidity Exposure to DextranPreserved 40° C. 50% Example sulfate platelets F1 + 2 N (number RH, 1week No. No. [%] [pmol/L] average) Pre 1.13 4 93 741 1 Post 4 97 1101 1Pre Uncoated — 2 332748 — Post PVC example Pre Clotting — 1 853981 —Post example

TABLE 18 Preserved platelets (%) and F1 + 2 (pmol/L) of PVC tubingcoated with dextran sulfates 4, 5 and 7 at 1.7M NaCl concentration -before and after exposure to increased temperature and humidity Exposureto Dextran Preserved 40° C. 50% Example sulfate platelets F1 + 2 N(number RH, 1 week No. No. [%] [pmol/L] average) Pre 1.15 4 97 2057 2Post 4 91 1435 1 Pre 1.22 5 90 3609 3 Post 5 91 1580 1 Pre 1.35 7 863112 1 Post 7 90 3301 1 Pre Uncoated — 1 619778 — Post PVC example PreClotting — 2 768361 — Post example

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

1. A process for the manufacture of a solid object having a surfacecomprising a layered coating of cationic and anionic polymer wherein theouter coating layer comprises an anticoagulant entity, comprising thesteps of: i) treating a surface of the solid object with a cationicpolymer; ii) treating the surface with an anionic polymer; iii)optionally repeating steps i) and ii) one or more times; iv) treatingthe surface with a cationic polymer; and v) treating the outermost layerof cationic polymer with an anticoagulant entity, thereby to covalentlyattach the anticoagulant entity to the outermost layer of cationicpolymer; wherein, the anionic polymer is characterized by having (a) atotal molecular weight of 650 kDa-10,000 kDa; and (b) a solution chargedensity of >4 μeq/g; and wherein, step ii) is carried out at a saltconcentration of 0.25 M-5.0 M.
 2. A process for the manufacture of asolid object according to claim 1, wherein the anionic polymer isdextran sulfate.
 3. A process for the manufacture of a solid objectaccording to claim 1, wherein the anionic polymer is characterized byhaving a total molecular weight of 750 kDa-10,000 kDa.
 4. (canceled) 5.A process for the manufacture of a solid object according to claim 1,wherein the anionic polymer is characterized by having a solution chargedensity of between >4 μeq/g and 7 μeq/g.
 6. A process for themanufacture of a solid object according to claim 1, wherein step ii) iscarried out at a salt concentration of 0.25 M-4.0 M.
 7. (canceled) 8.(canceled)
 9. A process for the manufacture of a solid object accordingto claim 1, wherein the salt is selected from the group consisting ofsodium chloride, sodium sulfate, sodium hydrogen phosphate and sodiumphosphate.
 10. A process for the manufacture of a solid object accordingto claim 9, wherein the salt is sodium chloride.
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. A process for the manufacture of a solidobject according to claim 1, wherein the cationic polymer of step i) isa polyamine.
 15. A process for the manufacture of a solid objectaccording to claim 1, wherein the cationic polymer of step iv) is apolyamine.
 16. (canceled)
 17. (canceled)
 18. A process for themanufacture of a solid object according to claim 1, wherein theanticoagulant entity is a heparin moiety.
 19. A process for themanufacture of a solid object according to claim 18, wherein the heparinmoiety is an end-point attached heparin moiety.
 20. A process for themanufacture of a solid object according to claim 19, wherein theend-point attached heparin moiety is connected through its reducing end.21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled) 25.(canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)30. (canceled)
 31. (canceled)
 32. (canceled)
 33. (canceled) 34.(canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled)39. (canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)
 43. A solidobject having a surface comprising a layered coating of cationic andanionic polymer, wherein the outer coating layer is a layer comprisingcationic polymer to which is covalently bound an anticoagulant entity;and wherein the anionic polymer is characterized by having (a) a totalmolecular weight of 650 kDa-10,000 kDa; and (b) a solution chargedensity of >4 μeq/g.
 44. A solid object according to claim 43, whereinthe anionic polymer is characterized by having a total molecular weightof 650 kDa-1,000 kDa.
 45. (canceled)
 46. (canceled)
 47. (canceled)
 48. Asolid object according to claim 43, wherein the anionic polymer isapplied to the surface at a salt concentration of 0.25 M-5.0 M.
 49. Asolid object according to claim 43, wherein the anionic polymer is apolymer comprising groups selected from —CO₂ ⁻, —SO₃ ⁻, —PO₃H⁻ and —PO₃²⁻.
 50. A solid object according to claim 49, wherein the anionicpolymer is a polymer comprising —SO₃ ⁻ groups.
 51. A solid objectaccording to claim 50, wherein the sulfur content of the anionic polymeris between 10% and 25% by weight of the anionic polymer.
 52. (canceled)53. (canceled)
 54. (canceled)
 55. (canceled)
 56. A solid object having asurface comprising a layered coating of cationic and anionic polymer,wherein the outer coating layer is a layer comprising cationic polymer;and wherein the anionic polymer is characterized by having (a) a totalmolecular weight of 650 kDa-10,000 kDa; and (b) a solution chargedensity of >4 μeq/g.
 57. (canceled)
 58. (canceled)
 59. (canceled) 60.(canceled)
 61. A solid object having a surface comprising a layeredcoating of cationic and anionic polymer, wherein the outer coating layeris a layer comprising anionic polymer; and wherein the anionic polymeris characterized by having (a) a total molecular weight of 650kDa-10,000 kDa; and (b) a solution charge density of >4 μeq/g. 62.(canceled)